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WO2015066189A2 - La modification de l'actine nucléaire par mical-2 régule la signalisation srf - Google Patents

La modification de l'actine nucléaire par mical-2 régule la signalisation srf Download PDF

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WO2015066189A2
WO2015066189A2 PCT/US2014/062915 US2014062915W WO2015066189A2 WO 2015066189 A2 WO2015066189 A2 WO 2015066189A2 US 2014062915 W US2014062915 W US 2014062915W WO 2015066189 A2 WO2015066189 A2 WO 2015066189A2
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mical
actin
mrtf
srf
cells
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WO2015066189A3 (fr
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Mark Lundquist
Samie R. Jaffrey
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Cornell University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • This disclosure relates to serum response factor (SRF) regulated pathways and more particularly to a specific modulator of SRF signaling.
  • SRF serum response factor
  • SRF serum response factor
  • GPCR G-protein coupled receptor
  • SRF-dependent gene transcription is modulated by SRF coactivators, including ternary complex factor (TCF) and myocardin-related transcription factor A (MRTF-A).
  • MRTF-A binds to SRF, forming a complex that influences SRF binding to the CArG box promoter element, which is found in SRF target genes.
  • SRF/MRTF-A-dependent gene transcription mediates diverse cellular processes including cellular migration, cancer cell metastasis, mammary myoepithelium development, and neurite formation.
  • SRF/MRTF-A-dependent gene transcription is induced when MRTF-A localizes to the nucleus.
  • MRTF-A is found in both the cytosol and the nucleus, but exhibits increased nuclear localization in response to various signaling pathways. The nuclear localization of
  • MRTF-A enables it to form complexes with SRF, resulting in transcription of genes that contain promoter elements that bind the SRF/MRTF-A complex.
  • SRF/MRTF-A-dependent gene transcription is highly influenced by the levels of nuclear MRTF-A.
  • MRTF-A localization is regulated by the availability of G-actin in the nucleus.
  • G-actin in the nucleus binds to MRTF-A, enabling it to be exported to the cytosol.
  • high levels of G-actin in the nucleus seen during serum deprivation lead to low levels of nuclear MRTF-A.
  • Activation of SRF/MRTF-A-dependent gene transcription occurs when signaling pathways reduce nuclear G-actin, which prevents MRTF-A export, and results in accumulation of MRTF-A in the nucleus.
  • SRF/MRTF-A-dependent gene transcription this mechanism is unlikely to mediate SRF/MRTF- A signaling in all cell types.
  • SRF/MRTF-A signaling regulates axon growth and other neuronal functions but stress fiber formation is not typically seen in neurons. Additional pathways that induce SRF/MRTF-A signaling and modulators of such putative signaling remain to be identified.
  • MICAL-2 a member of a family of atypical actin-regulatory proteins that induces redox-dependent depolymerization of nuclear actin, reduces nuclear G-actin levels.
  • MICAL-2 expression increases nuclear MRTF-A levels and mediates SRF/MRTF-A-dependent gene transcription elicited by nerve growth factor and serum.
  • MICAL-2 is a target of CCG-1423, a small molecule inhibitor of SRF/MRTF-A-dependent transcription that exhibits efficacy in various preclinical disease models.
  • the disclosure provides a method for identifying an agent which can inhibit SRF/MRTF-A pathway and/or which can inhibit activity of Axl comprising: contacting a test agents with MICAL-2; and determining if the activity of MICAL-2 is inhibited, wherein inhibition of MICAL-2 activity is an indication that the test agent inhibits the SRF/MRTF-A pathway and/or activity of Axl.
  • an agent or a plurality of agents may be tested simultaneously. The activity of MICAL-2 may be tested by any of the methods described herein.
  • an inhibition of MICAL-2 activity may be tested by detecting depolymerization of F-actin, conversion of F-actin to G-actin, a reduction in the induction of oxidative marks on actin, an increase in nuclear F-actin, a decrease in nuclear MRTF-A, a decrease in formation of SRF/MRTF-A complexes, a decrease in transcription of genes that are regulated by SRF/MRTF-A or any other action downstream of MICAL-2 or upstream of MICAL-2 action.
  • an agent or a plurality of test agents may be tested for their ability to enhance the activity of MICAL-2.
  • activation or enhancement of activity of MICAL-2 may be tested by detecting a reduction in
  • MICAL-2 is a nuclear protein that depolymerizes actin (A) Cherry-
  • MICAL-2 expressed in HEK293T cells is localized to the nucleus.
  • Cherry-tagged MICAL constructs were expressed in
  • Endogenous MICAL-2 and -3 are localized to the nucleus.
  • HEK293T cells were subjected to subcellular fractionation and endogenous MICAL-1, -2 and -3 were detected by western blot.
  • MEK1/2, VDAC, Calnexin, and PCNA immunoblotting was used to confirm the purity of the cytosolic (Cyto), mitochondrial (Mito), microsomal (Micro), and nuclear (Nuc) fractions, respectively.
  • Endogenous MICAL-1 was detected primarily in the cytosolic fraction, while MICAL-2 and -3 were detected in the nuclear fraction.
  • MICAL-2 and -3 each contain a putative bipartite NLS.
  • the domain structure of MICAL-2 is indicated above.
  • the enzymatic domain contains a GXGXXG, aspartate-glycine (DG), and a glycine-aspartate (GD) motif, which is the characteristic FAD- binding motif.
  • the F-actin binding CH domain and structural LIM domain are indicated.
  • the red box is the NLS domain in MICAL-2.
  • Comparison of the primary sequences of MICAL-1, -2 and -3 shows a potential bipartite NLS in MICAL-2 and MICAL-3 that is not found in MICAL- 1.
  • Shown in blue letters are the amino acids (RkR, krrrk, krrk) that constitute the NLS in MICAL-2 and -3. Shown in red are the amino acids (aaaaa, aaaa) that are mutated in the M2NLSMut and M3NLSMut. Uppercase letters are amino acids conserved in MICAL-1, -2 and -3.
  • MICAL-2 inhibits F-actin recombinant MICAL-1 redoxCH , MICAL-2 redoxCH and MICAL- 3 redoxCH j which comprise the enzymatic and CH domain, were incubated with pyrene-labeled actin.
  • This actin exhibits 7-12 times more fluorescence when it is in the polymeric F-actin than its monomeric G-actin form.
  • the fluorescence of pyrene-actin alone (black square) or pyrene- actin with MICAL-1 redoxCH (grey diamonds) remained relatively constant over time.
  • MICAL-1 redoxCH green circle
  • MICAL-2 redoxCH blue triangle
  • MICAL-3 redoxCH red triangle, inverted triangle
  • Statistical significance was determined by oneway analysis of variance (ANOVA) (***p ⁇ 0.0007) with Dunnett multiple comparison post- test. Each group was compared to F-actin alone. ***p ⁇ 0.0005, n > 12 per condition
  • HEK 293 cells were transfected with plasmids expressing GFP-MICAL-2 or either of two dominant-negative MICAL-2 constructs: GFP-MICAL-2CT, which lacks the enzymatic domain, and MICAL-2GV, which contains a point mutation in the second glycine of the FAD-binding domain (GXGXXG -> GXVXXG).
  • GFP-MICAL-2CT which lacks the enzymatic domain
  • MICAL-2GV which contains a point mutation in the second glycine of the FAD-binding domain
  • Expression of MICAL-2CT or MICAL-2 GV resulted in the appearance of prominent filaments throughout the nucleus (yellow arrows, arrows in the GFP row), as seen by labeling with Alexa 568-Phalloidin (red staining, blue arrows, arrows in the F-actin row).
  • F-actin polymers were only seen in transfected cells (indicated by the green fluorescence of the transfected MICAL-2 construct). The dominant negative constructs also colocalized with the F-actin (colocalization in yellow), suggesting that MICAL-2 binds and then disrupts F-actin polymers. Nuclei were labeled with DAPI. Scale bar represents 5 ⁇ .
  • MICAL-2 induces the nuclear localization of MRTF-A
  • A MICAL-2 expression is sufficient for increasing nuclear localization of MRTF-A under serum-starvation.
  • HEK293T cells were infected with lentivirus expressing either GFP or GFP-MICAL-2 for 24 hr and subsequently serum-starved in 0.3% FBS for 18 hr.
  • endogenous MRTF-A was localized primarily to the cytoplasm, as determined by anti-MRTF-A
  • PC 12 cells To determine if MICAL-2 mediates the nuclear localization of MRTF-A in response to NGF, PC 12 cells were infected with lentivirus expressing GFP (green) and either LacZ-specific shRNA or either of two MICAL-2-specific shRNA. NGF treatment induces nuclear localization of MRTF-A in LacZ shRNA-expressing PC 12 cells. However, PC 12 cells expressing either MICAL-2-specific shRNA (yellow (bright) arrows) showed markedly reduced NGF-induced nuclear localization of MRTF-A compared to uninfected cells (blue (dark) arrows). These data indicate that MICAL-2 is necessary for NGF-induced increase in the nuclear MRTF-A levels in PC12 cells. Nuclei are outlined by dotted white lines. Scale bar represents 10 ⁇ .
  • E The nuclear localization of MRTF-A in dissociated E14 DRG neurons cultured in the presence of NGF is dependent upon MICAL-2.
  • E14 DRG neurons are cultured in the presence of NGF, which is associated with prominent nuclear localization of MRTF-A (red).
  • Infection of DRG neurons with MICAL-2-specific shRNA results in a substantial depletion of MRTF-A from the nucleus.
  • Scale bar represents 5 ⁇ .
  • Nuclear, enzymatically active MICAL-2 induces the SRF/MRTF-A transcriptional reporter in the absence of serum.
  • MICAL-2 induces SRF/MRTF-A signaling
  • HEK293T cells were co-transfected with pGL4.34[luc2p/srf-re], a reporter construct expressing luciferase under the control of a SRF/MRTF-A-dependent CArG box-containing promoter and the indicated MICAL-2 variants.
  • Serum-starved HEK293T (DMEM, 0.3% FBS) cells were cultured for 18 h, and then treated with either 0.3% FBS or 10% FBS for 6 h.
  • luciferase expression was minimal following treatment with 0.3% FBS, but was induced following treatment with 10% FBS.
  • MICAL-2 expression markedly increased luciferase expression in 0.3% FBS-treated cells. This effect was abolished in
  • HEK293T cells expressing catalytically inactive MICAL-2 mutants (MICAL-2GV and MICAL- 2CT), as well as a MICAL-2 mutant that does not localize to the nucleus (M2NLSMUT).
  • MICAL-2GV and MICAL- 2CT catalytically inactive MICAL-2 mutants
  • M2NLSMUT MICAL-2 mutant that does not localize to the nucleus
  • pGL4.33 is an SRF-dependent reporter vector that is activated by the MAPK/ERK signaling pathway and not the RhoA/MRTF-A pathway.
  • MICAL-2 did not affect basal or serum-stimulated expression of this reporter.
  • MICAL-2 selectively activates SRF/MRTF-A-dependent gene expression.
  • MICAL-2 but not MICAL-1 or MICAL-3, activates the SRF/MRTF-A reporter.
  • SRF/MRTF-A luciferase reporter was a general MICAL effect or specific to MICAL-2.
  • HEK293T cells were co-transfected with pGL4.34[luc2p/srf-re] and each of the three MICAL isoforms.
  • Serum-starved HEK293T cells (DMEM, 0.3% FBS) were cultured for 24 h, and then treated with either 0.3% FBS or 10% FBS for 6 h.
  • MRTF-A MRTF-A.
  • MRTF - ⁇ AD dominant-negative MRTF-A construct
  • GFP GFP- MICAL-2
  • Coexpression of MRTF-AATAD blocked MICAL-2-dependent induction of luciferase.
  • MICAL-2 is required for NGF-dependent neurite outgrowth in PC12 cells and DRG neurons (A) MICAL-2 is required for NGF-induced neurite outgrowth in PC 12 cells.
  • PC 12 cells were infected with lentivirus expressing GFP (green) and either LacZ-specific or MICAL-2-specific shRNA.
  • NGF treatment 50 ng/mL
  • 48 h resulted in prominent neurite outgrowth in LacZ shRNA control as measured by Alex568-phalloidin staining (red).
  • MICAL-2 is necessary for neurite growth in DRG neurons.
  • Rat El 4- 15 DRG neurons cultured in NGF were infected with lentivirus expressing either LacZ-specific or MICAL-2-specific shR A, and axon growth rate was measured over a 60 min time period at DIV5. The position of the axon growth cone at the beginning of the experiment is indicated with the blue (dark) arrow, while the position at 60 min is indicated with a yellow (bright) arrow.
  • DRG neurons expressing MICAL-2-specific shRNA exhibit markedly reduced axonal growth rates. Scale bar represents 10 ⁇ .
  • FIG. 1 MICAL-2 regulates SRF/MRTF-A-dependent gene transcription in zebrafish (A,B) Representative control embryo derived from transgenic myl7:egfp reporter fish at 24 hpf, showing the size and position of a normal looping heart tube. By 48 hpf the control heart is fully looped.
  • the myl7:egfp transgene directs eGFP expression to myocardial cells and facilitates detection of alterations in heart morphology.
  • (C,D) mical2b is effectively knocked down in zebrafish.
  • morpholino treatment we examined cardiac-specific defects in morphant embryos.
  • the mical2b splice-blocking morphant embryo at 24 hpf shows a smaller heart tube that fails to loop normally and thus is positionally displaced and leads to a significant pericardial edema (PE).
  • PE pericardial edema
  • the morphant heart tube is linear and dysmorphic.
  • the representative embryonic hearts shown in F and G are for embryos injected with 2 or 4 ng of the splice-blocking morpholino, respectively (n>50).
  • MICAL-2 regulates nuclear actin independent of RhoA MICAL-2 induces the SRF/MRTF-A reporter in a ROCK- and RhoA-independent manner.
  • MICAL-2 induces SRF/MRTF-A-dependent gene transcription by activating RhoA.
  • RhoA and ROCK inhibitors we monitored the effect of RhoA and ROCK inhibitors on MICAL-2 -dependent induction of the SRF/MRTF-A reporter.
  • HEK293T cells were co-transfected with pGL4.34[luc2p/srf-re], a reporter construct expressing luciferase under the control of a SRF/MRTF-A-dependent CArG box-containing promoter and either GFP or GFP-MICAL-2.
  • HEK293T cells were treated with either 2 ⁇ g/mL C3 -transferase, a RhoA inhibitor, or 100 ⁇ Y27632, a ROCK inhibitor, along with either serum-starvation (0.3% FBS) or serum-stimulation (10% FBS).
  • serum-starvation (0.3% FBS)
  • serum-stimulation 10% FBS.
  • treatment with either inhibitor reduced luciferase expression induced by serum-stimulation.
  • these inhibitors did not reduce luciferase expression induced by MICAL-2 expression.
  • MICAL-2 does not induce SRF/MRTF-A signaling by inducing Rho A/ROCK.
  • Statistical significance was determined by ANOVA (***p ⁇ 0.0001) with Dunnett multiple comparison post-test. Stimulated HEK293T cells expressing GFP were compared to GFP-expressing cells stimulated in the presence of Rho-inhibitors.
  • MICAL-2 induces nuclear MRTF-A in a ROCK- and RhoA-independent manner.
  • RhoA nuclear MRTF-A accumulation by activating RhoA
  • HEK293T cells were infected with either GFP or GFP-MICAL-2.
  • HEK293T cells were treated with either 2 ⁇ g/mL C3-transferase, a RhoA inhibitor, or 100 ⁇ Y27632, a ROCK inhibitor, along with either serum-starvation (0.3% FBS) or serum-stimulation (10% FBS). Neither of these inhibitors reduced nuclear MRTF-A induced by MICAL-2 expression. Thus, MICAL-2 does not induce SRF/MRTF-A signaling by inducing RhoA/ROCK. Statistical was significance determined by ANOVA (***p ⁇ 0.0001) with Dunnett multiple comparison post-test. [0038] (C) MICAL-2 expression decreases the nuclear to cytosolic ratio of G-actin in
  • HEK293T cells In Figures 2A and 2B we demonstrate that MICAL-2 expression in HEK293T cells can increase the nuclear: cytosolic MRTF-A ratio in the absence of serum. To determine if this effect could be due to MICAL-2-dependent changes in nuclear G-actin, we monitored the effect of MICAL-2 expression on nuclear G-actin.
  • HEK293T cells were infected with lentivirus expressing either GFP or GFP-MICAL-2 for 24 hr and then serum-starved for 18 hr. In control GFP-expressing cells, G-actin is readily detectable in the nucleus, as measured by DNAse I staining (red). Serum stimulation significantly reduced the amount of G-actin in the nucleus.
  • GFP-MICAL-2 expression significantly reduced the levels of G-actin in the nucleus, even in the absence of serum-stimulation. These data show that MICAL-2 expression reduces nuclear G- actin levels. Nuclei were labeled with DAPI. Scale bar represents 5 ⁇ .
  • GFP-actin M44L When overexpressed, GFP-actin M44L is enriched in the nucleus when compared to wild-type GFP-Actin. MICAL-2 (red) coexpression further decreases the levels of nuclear wild- type GFP-actin (green) while having no effect on the nuclear levels of GFP-actin M44L. The nucleus is stained with DAPI (blue). Scale bar represents 5 ⁇ .
  • HEK293T cells were transfected with pGL4.34[luc2p/srf-re], a reporter construct expressing luciferase under the control of a SRF/MRTF-A-dependent CArG box-containing promoter.
  • Transfected cells were serum-starved for 18 hr and then treated with either serum-starvation media (DMEM, 0.3% FBS) or serum-stimulation media (DMEM, 10% FBS) for 6 hr.
  • DMEM serum-starvation media
  • DMEM serum-stimulation media
  • 10% FBS resulted in marked increase in luciferase expression in control cells, and MICAL-2 expression was sufficient to stimulate luciferase activity in the absence of serum.
  • CCG-1423 inhibits MICAL-2-induced reporter expression.
  • Statistical significance determined by ANOVA (***p ⁇ 0.0001) with Dunnett multiple comparison post-test. Stimulated HEK 293T cells expressing GFP were compared to all other conditions. ***p ⁇ 0.0005, n 24 per condition.
  • CCG-1423 exhibits concentration-dependent thermal destabilization of MICAL2-EN.
  • Recombinant MICAL2-EN which comprises the enzymatic domain of MICAL- 2
  • CCG-1423 was incubated with increasing concentrations of CCG-1423, or the control compound, CCG- 100594, and the T m was calculated in a thermal denaturation assay.
  • Incubation of CCG-1423 exhibited thermal destabilization of MICAL-2 with an IC 50 of 3.8 ⁇ and Hill coefficient -1.1. The Hill coefficient was calculated using a three parameter fitting procedure with the top constrained to zero.
  • CCG-100594 was not significantly different from DMSO at any concentration tested.
  • CCG-1423 inhibits MICAL-2 enzymatic activity.
  • MICAL-2 activity was monitored using an NADPH consumption assay.
  • MICAL-2 was incubated with either 5 ⁇ CCG- 1423 or the inactive control compound CCG-100594 in the presence of 2 ⁇ F-actin and 10 ⁇ NADPH. Consumption of NADPH was measured by the loss of absorbance at 365 nM.
  • Figure 8 related to Figure 1. Development of tools for characterization of the
  • Antibodies targeting the enzymatic domains of MICAL-1, -2, and -3 were affinity purified from immunized rabbit serum. Each purified antibody was then validated to confirm that it recognizes its respective MICAL isoform by western blot. For each blot, the first lane contained HEK293T lysate overexpressing GST while the second lane contained a HEK293T lysate overexpressing one of the three GST-MICAL isoforms. Each purified antibody recognizes a band corresponding to its overexpressed isoform in HEK293T cells.
  • Endogenous MICAL-2 is a nuclear protein, as measured by
  • MICAL-2 MICAL-2. After 4 days the cells were fixed and processed for immunofluorescence. Purified MICAL-2 antibody (green, left col.) exhibited a fluorescence signal that localized to the nucleus (blue, right col.) in shRNA LacZ-expressing cells. This signal was greatly diminished in MICAL-2 knockdown cells. These data (1) confirm the suitability of this antibody for immunofluorescence applications; and (2) demonstrate that endogenous MICAL-2 is localized to the nucleus. Neither purified anti -MICAL- 1 nor purified anti-MICAL-3 antibodies were suitable for immunofluorescence (data not shown). Scale bar represents 12 ⁇ .
  • MICAL-2 is a nuclear protein in multiple cell types.
  • endogenous MICAL-2 is a nuclear protein in multiple cell types.
  • COS7 and HeLa cells were fixed and processed for immunofluorescence using the antibodies described in A and C.
  • MICAL-2 immunofluorescence (green) colocalized with DAPI (blue) in both cell types. As is typically seen in most cells, no nuclear actin was seen by phalloidin staining (red). Scale bar represents 30 nm.
  • MICAL-2 constructs MICAL-2 CT and MICAL-2GV, induce the formation of F-actin filaments in the nucleus in HEK293T cells.
  • F-actin filament phenotype is not limited to HEK293T cells.
  • COS7, HeLa and differentiated PC12 cells were infected with a lentivirus expressing MICAL-2CT and fixed in PBS/4%PFA for immunofluorescence.
  • MICAL-2CT green, labeled with yellow arrows
  • F-actin polymers throughout the nucleus
  • Alexa Fluor 568- Phalloidin red, labeled with red arrows
  • the dominant-negative constructs also colocalized with the F-actin (colocalization in yellow), suggesting that the inactive form binds to F-actin filaments but was unable to disrupt them.
  • Nuclei were labeled with DAPI. Scale bar represents 5 ⁇ .
  • MICAL-2 knockdown induces nuclear F-actin filaments as visualized by phalloidin staining.
  • NIH3T3 cells were infected with a virus expressing shRNA targeting either LacZ or one of two shRNAs targeting MICAL-2.
  • Knockdown of MICAL-2 induced the formation of nuclear filaments similar to those seen with the expression of MICAL-2 dominant negative constructs ( Figure 9A). The nucleus is stained with DAPI (blue). Scale bar represents 5 ⁇ .
  • NLS-GFP-Lifeact is a F-actin binding protein that enables the imaging of F- actin in living cells. This protein is targeted to the nucleus by the inclusion of a NLS sequence. This system for imaging F-actin is particularly useful since the imaging is performed in living cells. Imaging F-actin in live cells is important since the integrity of F-actin can be
  • NLS-GFP-Lifeact construct was coexpressed in NIH3T3 cells with a vector expressing both mCherry and a LacZ- specific shRNA or one of two MICAL-2-specific shRNAs. Knockdown of MICAL-2 induced the formation of nuclear filaments detectable by Lifeact (green) similar to those seen with the expression of MICAL-2 dominant negative constructs ( Figure 9A). These data suggest that MICAL-2 normally inhibits F-actin formation in nuclei. The nucleus is stained with DAPI (blue). Scale bar represents 5 ⁇ .
  • MICAL-2 levels do not affect nuclear area. We looked if MICAL-2 could affect nuclear size. HEK293T cells expressing either of two MICAL-2-specific shRNAs, or GFP-MICAL-2 had no significant difference in size compared to control transfected cells. This indicates that MICAL-2 does not play a role in determining the size of cell nuclei. Statistical significance determined by ANOVA (ns). n > 40 per condition.
  • (L-0) MICAL-2CT induced actin filaments are within the nucleus.
  • a 3D volume was created from the images presented in (B). In the lower left hand corner of each panel we show the orientation of the axes relative to Figure 8L.
  • Lamin-A/C staining (green) labels the inner nuclear membrane.
  • a dotted line within the lamin-A/C stain demonstrates that the MICAL-2CT (red) induced filaments are internal and only contact the lamin-A/C staining at one point (blue arrow).
  • the nucleus is stained with DAPI (blue). Shown is the same nucleus from different perspectives. Scale bar represents 10 ⁇ .
  • MICAL-2 might be regulated by serum-activated signaling pathways. To test this, we asked if the phosphorylation status of MICAL-2 was affected by serum treatment.
  • HEK293T cells transfected with GST-MICAL-2 were serum starved overnight and then either treated with serum-free media or 20% FBS for 30 min.
  • GST-MICAL-2 was then purified on glutathione magnetic beads. Purified MICAL-2 was then assayed by western blot with antibodies against either GST (to measure total pulled down GST-MICAL-2) or phosphothreonine. Serum stimulation markedly increased the level of phosphothreonine in GST-MICAL-2. Experiments were also performed using anti-phosphoserine antibodies, but showed no effect of serum (data not shown).
  • P- threonine/GST ratio is the anti-phosphothreonine antibody signal for each condition divided by the anti-GST antibody signal.
  • Signal intensity was measured on a Biorad ChemiDoc MP system based on the fluorescence of the Cy3 or Cy5 secondary antibody. Statistical significance determined by student t-test. *p ⁇ 0.05, n > 4 per condition.
  • HEK293T cells To determine if MICAL-2 mediates the nuclear localization of MRTF-A in response to serum, HEK293T cells were infected with lentivirus expressing both GFP and either of two MICAL-2-specific shRNA. Stimulation with 2.0% FBS for 10 min after serum- starvation induces nuclear localization of MRTF-A in LacZ shRNA-expressing HEK293T cells. However, HEK293T cells expressing MICAL-2-specific shRNA showed markedly delayed serum-induced nuclear localization of MRTF-A compared to control cells. Nuclei are outlined by dotted white lines. Scale bar represents 10 ⁇ . [0063] (D) Quantification of results in (A). Statistical significance determined by
  • HEK293T cells One possible mechanism by which MICAL-2 could induce SRF/MRTF-A- dependent gene expression is by inducing the expression of MRTF-A.
  • HEK293T cells were infected with lentivirus expressing either GFP or GFP-MICAL-2 for 24 hr. Cells were then serum-starved for 18 hr at which point they were either serum-starved or serum- stimulated for 1 hr before harvesting for western blot. As shown, there were no substantial alterations in MRTF-A expression levels following MICAL-2 expression. This argues against the idea that MICAL-2 directly induces an increase in MRTF-A expression levels.
  • panel I and K we show that MICAL-2 and serum, respectively, does not induce stress fibers in HEK293T cells. However, we wanted a positive control to show that we can indeed detect stress fibers if they form.
  • NIH3T3 cells readily form stress fibers in response to serum.
  • MICAL-2 does not induce stress fibers in HEK293T cells.
  • serum-starved cells expressing GFP green
  • little to no F-actin stress fibers red, labeled with yellow arrows
  • Expression of GFP- MICAL-2 does not induce stress fibers in HEK293T cells, indicating MICAL-2 induces nuclear MRTF-A through a separate mechanism.
  • Scale bar represents 5 ⁇ .
  • Phalloidin signal in the cytosol of GFP-infected cells was compared to phalloidin signal in knockdown or
  • MICAL-2 overexpression does not affect transcript levels of MICAL family members. It was conceivable that MICAL-2 may have activated SRF/MRTF-A signaling through the induction or suppression of the transcription of other MICAL family members. To assay this possibility we analyzed the transcript levels of MICALs-1, -2, and -3 as well as MICALLs-1 and -2 using qRT-PCR after expression of either GFP or GFP-MICAL-2.
  • MICAL-2 expression lead to greatly increased transcript levels detected by qRT- PCR with MICAL-2-specific primers; however there was no change detected in the transcript levels of other MICAL isoforms.
  • MICAL-2 knockdown does not affect transcript levels of MICAL family members.
  • panel (A) we asked if MICAL-2 overexpression affects the transcript levels of MICAL family members.
  • MICAL-2 shRNA expression greatly decreased MICAL-2 transcript levels; however, there was no change detected in the other MICAL isoforms.
  • Statistical significance determined by ANOVA (***p ⁇ 0.0005) with Dunnett multiple comparison post-test. ***p ⁇ 0.0005 n > 3 per condition.
  • HEK293T cells SRF/MRTF-A-dependent genes in HEK293T cells.
  • HEK293T cells were plated at 100,000 cells/mL in 12 well plates and infected with either a GFP or GFP-MICAL-2 lentivrus and grown in serum-containing media for 24 hr. After 24 hr, serum was removed and cells were serum- starved for 18 hr, at which point the cells were harvested and processed for qRT-PCR.
  • the fold change in the SRF/MRTF-A-dependent genes Acta2, Cyr61, SRF, and VCL relative to housekeeping gene GAPDH was then compared between the GFP and GFP-MICAL-2 samples.
  • GFP-MICAL-2 significantly increases levels of Acta2, Cyr61, SRF, and VCL when compared to GFP expressing cells.
  • Expression levels of MRTF-A-independent SRF-dependent genes TSPl, PTGS1, and Egr2 did not differ significantly between GFP and GFP-MICAL-2 infected samples.
  • MICAL-2 can increase the expression of SRF/MRTF-A - dependent genes in the absence of serum stimulation.
  • Statistical significance was determined using ANOVA (*p ⁇ 0.02) with Dunnett multiple comparison post-test. Serum-starved HEK293T cells expressing MICAL-2 were compared to serum-starved GFP-expressing HEK293T cells. *p ⁇ 0.01, n > 4 per condition.
  • SRF/MRTF-A dependent genes in HEK293T cells Here we examine whether MICAL-2 is required for the induction of SRF/MRTF-A genes.
  • HEK293T cells infected with either of two shRNA lentiviruses targeting MICAL-2 were plated at 100,000 cells/well in 12 well plates. Cells were then serum-starved for 18 hr, at which point the media was removed and replaced with either serum-starved or serum-containing media for 2 hr. The fold change in gene expression was calculated by comparing the change in transcript levels between serum-starved and serum stimulated conditions.
  • HEK293T cells Statistical significance was determined by ANOVA (*p ⁇ 0.001) with Dunnett multiple comparison post-test.
  • HEK293T cells expressing shRNA LacZ were compared to HEK293T cells expressing one of two shRNA constructs targeting MICAL-2 expression. *p ⁇ 0.01, n > 4 per condition.
  • MICAL-2 overexpression induces the expression of SRF/MRTF-A- dependent genes in serum-starved PC 12 cells.
  • PC 12 cells were plated at 100,000 cells/mL in 12 well plates and infected with either a GFP or GFP-MICAL-2 lentivirus and grown in growth media for 24 hr. After 24 hr, growth media was removed and cells were grown in differentiation media without NGF for 18 hr.
  • the fold change in the SRF/MRTF-A- dependent genes Acta2, Cyr61, and SRF and relative to housekeeping gene GAPDH was then compared between the GFP and GFP-MICAL-expressing cells.
  • GFP-MICAL-2 significantly increases levels of Acta2, Cyr61, and SRF when compared to GFP expressing cells.
  • Expression levels of MRTF-A-independent SRF-dependent genes TSPl, PTGS1, and Egr2 did not differ significantly between GFP and GFP-MICAL-2-expressing cells.
  • MICAL- 2 can increase the expression of SRF/MRTF-A-dependent genes in the absence of stimulation.
  • Statistical significance was determined by ANOVA (*p ⁇ 0.01) with Dunnett multiple comparison post-test. Serum-starved PC 12 cells expressing MICAL-2 were compared to serum- starved GFP-expressing PC12 cells. *p ⁇ 0.01, n > 4 per condition.
  • PC 12 cells infected with either of two shRNA lentiviruses targeting MICAL-2 were plated at 100,000 cells/well in 12 well plates. Cells were then stimulated with 100 ng/mL NGF in serum-starved media at 24 hr and 72 hr post plating, before being harvested for qRT-PCR at 72 hr. Comparison between control knockdown and MICAL-2 knockdown demonstrates a significant decrease in NGF- induced expression of Acta2, Cyr61, VCL, and SRF relative to GAPDH.
  • SRF/MRTF-A dependent genes SRF/MRTF-A dependent genes.
  • MICAL-2 expression is required for the expression of SRF/MRTF-A-dependent genes seen in DRG neurons that are cultured in NGF- containing media.
  • Rat E14-15 DRG neurons were infected with lentivirus expressing control or MICAL-2-specific shRNA upon plating and harvested on DIV4.
  • Comparison between control knockdown and MICAL-2 knockdown demonstrates a significant decrease in NGF- induced expression of Acta2, Cyr61, VCL, and SRF relative to GAPDH.
  • Expression levels of MRTF-A- independent SRF-dependent genes TSP1, PTGS1, and Egr2 did not differ significantly between control and MICAL-2 shRNA samples.
  • Figure 5 we target the expression ⁇ micaUb in zebrafish using a splice-blocking morpholino.
  • this figure we detail how we targeted the 3' splicing junction between Exon 3 and Exon 4, which prevents the junction from being used and prevents the inclusion of Exon 3.
  • the frame shift induced by skipping Exon 3 introduces a premature stop codon in the mRNA, preventing the production of full-length mical2b.
  • mical2b morpholino targets the splice junction between exons 3 and 4.
  • Primers for detecting micaUb expression flanked the Exon 3 and exon 4 junction, while primers detecting mical2b-mutant flanked the Exon 2 and Exon 4 junction. It can be seen that the morpholino treatment decreases expression of wild-type micaUb mRNA and increases expression of the misspliced mRNA. Actin expression was monitored as a loading control.
  • mical2b knockdown were due to decreased mical2b expression and not off-target effects by utilizing a translation-blocking morpholino.
  • the schematic shows how the morpholino blocks recognition of the start codon.
  • FIG. 6E and 6F we demonstrated that MICAL-2 expression leads to decreased DNase I staining in the nucleus in HEK293T cells.
  • MICAL-2 expression leads to decreased DNase I staining in the nucleus in HEK293T cells.
  • this effect of MICAL-2 on G-actin in the nucleus is also seen in PC 12 cells.
  • PC 12 cells were infected with lentivirus expressing either GFP or GFP-MICAL-2 for 24 hr and then cultured in differentiation media (RPMI, 1% horse serum, 0.5% FBS, IX Pen/Strep) containing vehicle or 50 ng/mL NGF for 48 hr.
  • differentiation media RPMI, 1% horse serum, 0.5% FBS, IX Pen/Strep
  • G-actin is readily detectable in the nucleus, as measured by DNAse I staining (red).
  • NGF treatment significantly reduced the amount of G-actin in the nucleus.
  • GFP-MICAL-2 expression significantly reduced the levels of G-actin in the nucleus, even in the absence of NGF-stimulation.
  • Dotted white lines outline nuclei. Scale bar represents 10 ⁇ .
  • PC 12 cells In this figure we demonstrate that MICAL-2 is necessary for the decrease in nuclear G-actin levels following NGF stimulation in PC 12 cells.
  • PC 12 cells were infected with lentivirus expressing GFP (green) and either an shRNA targeting LacZ or MICAL-2. Cells were treated with 50 ng/ml NGF as in A, and G-actin levels in the nucleus were measure by DNAse I staining (red). Knockdown of MICAL-2 prevented the decrease in DNase I staining induced by NGF stimulation, which demonstrates that MICAL-2 is necessary for the decrease in nuclear G- actin levels in PC12 cells after NGF stimulation. Dotted white lines outline nuclei. Scale bar represents 10 ⁇ .
  • E MICAL-2 knockdown in DRG neurons increases nuclear G-actin levels.
  • MICAL-2 regulates nuclear G-actin levels in DRG neurons.
  • E14 DRG neurons were infected with either a lentivirus expressing LacZ- or MICAL-2-specific shRNA and cultured in the presence of NGF for three days. G-actin was detected by staining with DNAse I (red).
  • Knockdown (detected by GFP expression from the lentiviral construct, green) of MICAL- 2 increased G-actin levels in the nucleus as compared to control neurons.
  • Regions of interest were generated within NIS-elements software.
  • the Pearson colocalization coefficient which measures the fraction of green fluorescent protein (GFP) pixels that are also positive for phalloidin, was performed on randomly selected GFP-positive cell images (n > 20) using the NIS elements imaging software.
  • a colocalization coefficient of 1 indicates complete colocalization, whereas 0 means no colocalization.
  • Statistical significance determined by ANOVA (***p ⁇ 0.0001) with Dunnett multiple comparison post-test.
  • HEK293T cells expressing wild-type GFP-actin were compared to all other conditions. **p ⁇ 0.005, n > 30 per condition.
  • MICAL- 1 We used this buffer to conform to previous published MICAL assay conditions. However, to more accurately quantify the K m for NADPH, we performed an enzyme assay using buffer conditions that more accurately reflect the physiologic ion concentrations seen in the cytosol. The enzyme velocity of recombinant MICAL-2 was measured as a function of NADPH in 50 niM NaH 2 P0 4 , 100 niM KC1, 10 niM NaCl, 2 mM MgCl 2 , pH 7.4. MICAL-2 redoxLH was incubated with the indicated concentrations of NADPH and consumption of NADPH was measured by the loss of absorbance at 365 nM on a SpectraMax M2e (Molecular Devices). These results indicate that MICAL-2 uses NADPH with a K m of -153 ⁇ .
  • FIG. 15 AXL drives SRF/MRTF-A activity.
  • AXL inhibitor R428 inhibits SRF/MRTF-A-dependent luciferase activity in low micromolar concentrations over 4 hours.
  • AXL knockdown blocks GAS6-induced nuclear MRTF-A translocation into the nucleus, which is potentiated by GFP-AXL overexpression.
  • This disclosure provides a novel mechanism that regulates SRF/MRTF-A- dependent gene expression which involves depolymerization of nuclear actin by MICAL-2, a member of a family of recently described atypical actin-regulatory proteins.
  • MICAL-2 is homologous to MICAL-1, an enzyme that binds to F-actin in the cytosol and triggers its depolymerization through a redox modification of methionine.
  • MICAL-2 is enriched in the nucleus, and induces depolymerization of F-actin in the nucleus.
  • Expression of MICAL-2 leads to depletion of nuclear actin, resulting in nuclear retention of MRTF-A, and subsequent activation of SRF/MRTF-A-dependent gene transcription.
  • MICAL-2 is required for SRF/MRTF-A-dependent gene expression in several cell types, and mediates NGF-dependent neurite growth in neuronal cells. Furthermore, we find that CCG-1423, a small molecule SRF/MRTF-A pathway inhibitor that exhibits efficacy in various preclinical disease models, directly binds MICAL-2 and inhibits its activity. Together these data show that SRF/MRTF-A signaling is regulated by MICAL-2-dependent redox regulation of nuclear actin.
  • this disclosure provides a method for identifying modulators of
  • the modulators may be activators or inhibitors of the MICAL-2 activated SRF/MRTF-A pathway.
  • the method comprises testing potential agents for their ability to inhibit or enhance the activity of MICAL-2.
  • the agent may modulate the pathway upstream or downstream of MICAL-2 activation.
  • the screening method may involve evaluating the effect of agents on steps that are upstream or downstream of activation of MICAL-2 in the MICAL-2 activated SRF/MRTF-A pathway.
  • potential candidates may be tested for their effects on
  • MICAL-2 directly. These effects include binding to MICAL-2, or interference or enhancement of MICAL-2 enzymatic activity.
  • the effect of candidate agents is tested on MICAL-2 's ability to induce actin depolymerization in vitro, or inducing nuclear actin polymerization and/or depletion, or change in nuclear or cytosolic actin content or nuclear vs. cytosolic actin ratio.
  • the effect of candidate agents are tested on MICAL-2's ability to mediate nuclear localization of MRTF-A, whether by itself or in response to other agents (such as NGF).
  • the effects of candidate agents are tested on MICAL-2's ability to induce SRF/MRTF-A dependent gene expression, whether by itself of in response to inducement by agents such as serum and NGF.
  • candidate agents may be tested for effect on one or more steps involved in the MICAL-2 activation of SRF/MRTF-A or on events downstream of the activation of SRF/MRTF-A as described herein.
  • the screening assays may be carried out by in vitro using purified or recombinant
  • MICAL-2 or in cells in vitro, or may be carried out in animal models. Any type of cells in culture may be used. For example, any cell line or primary or secondary cultures may be used. Examples of suitable cells include HEK293T, COS7, NIH3T3 as well as HeLa cells. Primary cultures include DRG neurons, fibroblasts and the like.
  • the cells may be modified cells.
  • the cells may be engineered to overexpress MICAL-2 or may be naturally occurring overexpressors of MICAL-2.
  • the cells may be overexpressing SRF reporters or maybe engineered to overexpress SRF reporters.
  • the cells in culture can be maintained by using routine cell culture reagents and procedures.
  • the assays may be carried out in animals including vertebrates, such as zebrafish.
  • An effect on the expression or activity of MICAL-2 may be determined by evaluating one or more of the effects of activation of MICAL-2 as described herein. For example, depolymerization of actin or the SRF/MRTF-A pathway be evaluated.
  • the screening method is based on enzymatic assays to directly determine the activity of MICAL-2.
  • luciferase or fluorescent assays are used to determine the effects of the activity of MICAL-2.
  • a method for identifying an agent that inhibits one or more points in the SRF/MRTF-A pathway comprising: a) contacting a test agent with MICAL-2; and b) determining if the activity of MICAL-2 is inhibited, wherein inhibition of MICAL-2 activity is an indication that the test agent can inhibit the SRF/MRTF-A pathway.
  • the test agent may be contacted with isolated or recombinant MICAL-2, or the test agent may be contacted with cells that have MICAL-2, or have been engineered to overexpress MICAL-2.
  • the test agent may be administered to a living organism at any stage of development. For example, the test agent may be administered to a vertebrate during embryonic development.
  • Test agents having a desired level of effect compared to a control may be selected from the screening tests described herein.
  • test agents are identified that have an statistically significant effect over a negative control.
  • test agents are identified that have at least 5% or 10% effect over a negative control.
  • a negative control may be a sample in which an agent known not to have an effect is used or may be one that does not have the test agent.
  • a positive control that is known to have an effect may be used.
  • the positive control is CCG-1423.
  • test agents are identified that have at least the effect of CCG-1423.
  • test agents are identified that have up to 10%, 15% or 20% less effect than CCG-1423.
  • MICAL-2 activity or that are enhancers of MICAL-2 activity may be identified.
  • any of the activities or tests described herein may be used with a negative control that does not have the test agent to see if the activity that is known to be induced by MICAL-2 is increased or decreased. Based on that observation, the test agent may be identified as being an inhibitor, an activator or not effective. In one embodiment, if an agent has a statistically significant effect, or has at least a 5% or 10% effect (whether to increase or to decrease the effect of MICAL-2) it is considered to be a modulator of MICAL-2 activity.
  • positive controls may also be used which contain an agent known to have the effect that is desired in the test agent.
  • a positive control for testing for an inhibitor of MICAL-2 can be an agent which is known to inhibit the activity of MICAL-2 as determined by any of the methods disclosed herein or any other known method.
  • a positive control for testing for an enhancer of MICAL-2 can be an agent which is known to enhance the activity of MICAL-2 as determined by any of the methods disclosed herein or any other known method.
  • the effect of candidate agent is tested on the enzymatic activity of MICAL-2.
  • the effect of agents may be evaluated on the ability of
  • MICAL-2 to induce actin depolymerization.
  • This assay is based on the ability of MICAL-2 to depolymerize F-actin specifically in the presence of NADPH.
  • MICAL-2, F-actin and NADPH may be combined and the enzymatic activity of MICAL-2 detected as conversion of F-actin to G-actin.
  • the conversion of F-actin to G-actin can be monitored by using dyes or detectably labeled antibodies that bind differentially to F-actin and G-actin.
  • pyrene-actin or pyrene-conjugated antibodies (excitation:365nm emission:407nm) to actin may be used to assay the polymerization state of actin.
  • MICAL-2 and NADPH are added in the presence or absence of one or more test agents, and the fluorescence intensity is monitored over time. Fluorescence intensity may be measured by standard equipment.
  • test agents depolymerization by MICAL-2 can be compared to a control which does not have the test agent.
  • the results of test agents may be compared to the results obtained with CCG-1423.
  • the MICAL-2 activity is monitored by measuring the oxidation state of actin (or other proteins).
  • MICAL-2 induces oxidative marks on actin, such as methionine sulfoxides, and these marks can be measured as they are indicators of a change in the polymerization state of actin.
  • incubation of MICAL-2 in the presence and absence of test agents, with a protein (such as actin) may be carried out, followed by monitoring of the appearance of oxidative marks on the protein, such as by Western blot using antibody selective for those altered proteins, mass spectrometry, or other techniques that can detect changes in protein composition, to be used as an indicator of MICAL-2 activity.
  • An increase in oxidative marks compared to negative control is indicative of or enhanced MICAL-2 activity, whereas a decrease in oxidative marks is indicative of inhibition of MICAL-2 activity.
  • MICAL-2 activity may be detected by monitoring the conversion of NADPH to NADP.
  • MICAL-2 induced depolymerization of F-actin is monitored by the conversion of NADPH to NADP (excitation:360 emission:455 absorption: 360). Fluorescence intensity may be measured using standard equipment (such as on a SpectraMax M2e) at 455 nm with an excitation wavelength of 360 nm. The absorbance of NADPH may be subsequently read at 365 nm.
  • thermofluor assay may be used to evaluate MICAL-2 enzymatic activity.
  • the thermofluor assay uses any dye whose fluorescence increases when bound to the hydrophobic residues of proteins to determine the melting temperature of a specific protein. As the temperature increases the protein denatures, exposing more hydrophobic residues and increasing the fluorescence. When drugs bind to proteins they increase the stability of the protein and shift the melting temperature. Therefore the thermofluor assay can indicate which drugs specifically bind to proteins. For example, thermal denaturation of the enzymatic domain of MICAL2 may be monitored as an increase in dye (such as l-anilinonaphthalene-8- sulfonic acid (1,8 ANS)) fluorescence.
  • dye such as l-anilinonaphthalene-8- sulfonic acid (1,8 ANS)
  • inhibition of MICAL-2 activity is determined by detecting an increase in nuclear F-actin.
  • a test agent is contacted with a cell which has MICAL-2. Drugs that inhibit MICAL-2 activity can be expected to induce MICAL-2 bound F-actin filaments.
  • Candidate test agents may be contacted with cells in culture. After a suitable period of time, the cells may be processed to determine if nuclear F-actin content has increased. Nuclei may be isolated or histochemical or immunofluorescent techniques may be used to identify F-actin in the nucleus.
  • F-actin in the nucleus may be detected using dyes that specifically bind F-actin (such as Alexa 568-Phalloidin) or fluorescently labeled peptides that specifically bind to F-actin such as GFP-LifeAct may be used.
  • Lifeact is a sequence of 12 amino acids that specifically bind to F-actin and when attached to fluorescent proteins such as GFP can serve as an endogenously expressed marker for F-actin in cells.
  • a nuclear localized Lifeact construct recognized nuclear F-actin filaments when MICAL-2 was knocked down using shRNA constructs. This construct can be used to visualize nuclear F-actin induced by MICAL-2 inhibition by candidate agents.
  • a pathway comprises contacting cells with test agents and detecting the inhibition of MICAL-2 activity by detecting a reduction in the nuclear to cytosolic ratio of MRTF-A. This test is based on our observation that MICAL-2 activity increases the nuclear to cytosolic MRTF-A ratio.
  • the determination is carried out under serum-stimulated or MICAL-2 overexpressed conditions.
  • the comparison of cytosolic and nuclear MRTF- A is carried out by using primary antibodies to MRTF-A and then fluorescent secondary antibodies. Staining and imaging is done by routine methods.
  • SRF/MRTF-A regulated gene expression in the absence or presence of candidate agents is based on our observations that MICAL-2 activates SRF/MRTF-A-dependent gene transcription in the absence of serum and potentiate its activation in the presence of serum.
  • the activation of SRF/MRTF-A-dependent gene transcription is as measured with a luciferase protein downstream of the SRF/MRTF-A promoter region, may be used as an indicator of MICAL-2 activity.
  • a control MICAL-2 may be tested against the alternative SRF promoter, the SRF/TCF promoter.
  • Cells in culture can be transfected with the various expression vectors using standard transfection methods. After a suitable period of time, expression of the fluorescent proteins may be verified by fluorescence microscopy.
  • the effect on candidate agents may be tested on the expression of genes that are regulated by the SRF/MRTF-A pathway.
  • qPCR techniques may be used to test candidate agents for effects on the activity of MICAL-2 by screening elements downstream of SRF/MRTF-A pathway.
  • MICAL-2 activity regulates SRF/MRTF-A-dependent gene transcription, therefore inhibition of MICAL-2 activity with potential drug candidates should lead to lower expression of downstream SRF/MRTF-A target genes.
  • genes include serum response factor (SRF), Vinculin (VCL), beta integrin 1 (ITGBl), transforming growth factor beta 1 induced transcript 1 (TGFBIII), ⁇ -actin, cysteine-rich, angiogenic inducer 61 (CYR61), connective tissue growth factor (CTGF), four and a half LIM domains 2 (FHL2).
  • SRF serum response factor
  • VCL Vinculin
  • IGBl beta integrin 1
  • TGFBIII transforming growth factor beta 1 induced transcript 1
  • CYR61 transforming growth factor beta 1 induced transcript 1
  • cysteine-rich cysteine-rich
  • angiogenic inducer 61 angiogenic inducer 61
  • CTGF connective tissue growth factor
  • FHL2 half LIM domains 2
  • MICAL-2 activity assays may be carried out in vivo.
  • MICAL-2 knockdown through morpholinos is known to lead to severe cardiac phenotypes in zebrafish.
  • Suitability for MICAL-2 inhibitors in vertebrates may therefore be assessed by evaluating whether MICAL-2 inhibitors phenocopied the effects of the MICAL-2 morpholinos.
  • Zebrafish embryos may be injected with morpholinos at the 1 cell stage and allowed to develop until imaged.
  • Embryos may be processed for analysis for fluorescence imaging or qPCR at the desired developmental stage. The effect of test candidates may be assessed in comparison to the effects observed with the morpholinos.
  • candidate agents may be tested for their ability to inhibit
  • MICAL-2 regulation of SRF/MRTF-A pathway by testing for effects of the agents on steps upstream of MICAL-2.
  • candidate agents may be tested for their ability to interfere with Axl or its ligand Gas6.
  • a cell line can be treated with Gas6, a small molecule that mimics Gas6, a small molecule that binds to Axl and increases Axl signaling, a small molecule that activates the pathway and leads to increased Axl activity, and then a candidate agent can be tested for its ability to inhibit MICAL-2 regulation of SRF/MRTF-A pathway, by measuring the activity levels of MICAL-2, for example by purifying this protein using immunoprecipitation or other methods and quantifying its activity as described herein.
  • candidate molecules can be tested for their ability to induce the MICAL-2 pathways measured by the increase in SRF/MRTF-A signaling.
  • SRF/MRTF-A reporter constructs include the use of SRF/MRTF-A reporter constructs, measurement of endogenous gene expression for genes that contain SRF/MRTF-A response elements, the nuclear localization of MRTF-A, which is induced by MICAL-2, and other measures of MICAL-2 signaling activity.
  • this disclosure provides a method for identifying Axl inhibitors by detecting the effect of potential Axl inhibitor candidates on MICAL-2 activity by one or more methods disclosed herein.
  • the method comprises the steps of evaluating MICAL-2 activity after exposure to test agents.
  • the exposure may be in the form of contacting isolated or recombinant MICAL-2 in vitro, contacting cells in vitro, or administration to animals.
  • this disclosure also provides a method of inhibiting Axl pathway by using one or more inhibitors of MICAL-2 activity that are identified by the methods described herein.
  • one or more of the methods described herein may be used for determining the modulation of activity of MICAL-1 and/or MICAL-3 and therefore, the methods described herein may be used to identify the inhibitors or activators of MICAL- 1 or MICAL-3, in addition to identifying the inhibitors or activators of MICAL-2.
  • this disclosure provides a method for inhibiting the activity of
  • a method for inhibiting the formation of functional nuclear SRF/MRTF-A complexes by inhibiting the expression or activity of MICAL-2 comprises contacting a cell, in which inhibition of expression or activity of MICAL-2 is desired, with an inhibitor of MICAL-2.
  • the inhibitor of MICAL-2 expression or activity is a nucleic acid including interfering RNA (such as, for example, shRNA) or is a small molecule (such as, for example, CCG-1423).
  • the disclosure provides a method for inhibiting MICAL-2 activity by introducing into the cell an amount of CCG-1423, which is effective in inhibiting the expression or activity of MICAL-2.
  • the disclosure comprises inhibiting metastasis of cancer cells.
  • the method comprises administering to an individual a therapeutically effective dose of an inhibitor of MICAL-2.
  • the inhibitor of MICAL-2 is CCG-1423.
  • the inhibitor of MICAL-2 can be administered by any route including intravenous, intramuscular, intratumoral, transdermal or any other means which will deliver the agent or its effective metabolite to the site of action in an amount that will inhibit the metastasis of cancer cells.
  • the disclosure provides a method for promoting the formation of functional nuclear SRF/MRTF-A complexes in a cell by increasing the activity of MICAL-2 in the cell.
  • Increasing the formation of such complexes can be used for stimulating the growth and/or differentiation of neuritis (dendrites, axons, growth cones etc.) such as during nerve development or regeneration.
  • HEK293T, COS7, NIH3T3, and HeLa cells were cultured in DMEM containing
  • PC12 cells were cultured in RPMI containing 10% horse serum, 5% fetal bovine serum, glutamine and IX Pen- Strep.
  • DRG neurons were cultured in Neurobasal medium supplemented with B-27 (GIBCO), glutamine, 5-fluoro-2'-deoxyuridine (5-FdU, 10 ⁇ , Sigma) and GF (2.5 S, 50 ng/ml, Invitrogen).
  • HEK293T, COS7, NIH3T3, and HeLa cells were cultured in DMEM containing 10% fetal bovine serum.
  • PC 12 cells were cultured in DMEM containing 10% horse serum, 5% fetal bovine serum and pen-strep.
  • Dorsal root ganglion (DRG) neurons harvested from E14-15 rat embryos were dissected and cultured as previously described (Wu et al, 2005). Briefly, DRG neurons were dissected in L-15 and dissociated in TrypLE for 15 min before plating. Cells were grown in Neurobasal medium supplemented with B-27, glutamine, 5- fluoro-2'-deoxyuridine (5-FdU, 10 ⁇ , Sigma) and NGF (2.5 S, 50 ng/ml).
  • Luciferase levels were measured using ONE-glo (Promega) and with a Spectramax L luminometer (Molecular Devices). Further details are provided below.
  • HEK293T cells at 40-60% confluency were transfected with the various expression vectors using the calcium phosphate transfection method. Two days after transfection expression of the fluorescent proteins was verified by fluorescence microscopy. The cells were then trypsinized, counted, resuspended at a concentration of 5 x 105 cells/mL and 200 ⁇ ⁇ of each condition was plated in each of 12 wells in a 96-well plate. After 24 hr the cells were washed once with PBS and then serum-starved overnight (DMEM with 0.3% FBS).
  • each well was washed with PBS, then six of the 12 wells were serum-starved while the other six were serum-stimulated (DMEM with 10% FBS) for 6 hr.
  • DMEM serum-stimulated
  • each well was pretreated with the drug for 1 hr before addition of serum.
  • each well was rinsed with PBS and then 100 ⁇ ⁇ of One-Glo luciferase reagent and incubated in the dark at room temperature for 5 min. Luminescence was then read on a SpectraMax L luminometer (Molecular Devices).
  • PC 12 cells were infected with either overexpression or knockdown lentivirus and plated on 25 mm glass coverslips. PC 12 cells were then differentiated with 50 ng/mL as described previously (Geneste, 2002, The Journal of Cell Biology 157, 831-838; Wang et al, 2003, Proc.
  • PC 12 cells were plated at a density of 50,000 cells/well in a 12-well plate. 6 hr after plating cells were infected with either the LacZ control shRNA or one of two MICAL-2 knockdown shRNA described above. After three days, which leads to substantial knockdown of MICAL-2, cells were split onto PDL-coated glass coverslips at a concentration of 20,000 cells/mL in media (RPMI-1640, 10% horse serum, 5% FBS, IX pen-strep). 24 hr after plating the cells were washed with PBS and either serum-starved (DMEM, 0.3% FBS) or differentiated (DMEM, 0.3% FBS, 50 ⁇ g/mL NGF). 48 hr later the media was replaced with either fresh serum-starvation media or fresh differentiation media. After an additional 48 hr the media was replaced again for 15 min, at which point the slides were fixed as described below.
  • DMEM serum-starved
  • DMEM differentiated
  • Wildtype zebrafish (AB/Tu hybrid strain) were maintained as described Knoll and Nordheim, 2009, Trends Neurosci. 32, 432-442; Westerfield, 1993, The zebrafish book: A guide for the laboratory use of zebrafish (Brachydanio rerio).
  • Transgenic reporter strains tg(myl7:egfp) and myl7:actn3b-egfp were kindly provided by D. Yelon (UCSD). Morpholinos were purchased from Genetools, Inc. Sequences and experimental details are provided below.
  • Embryos were injected with morpholinos at the 1 cell stage and allowed to develop at 28.5C until imaged.
  • Morpholinos were titrated and injected in a volume of 2 nl using a PLI-100 Pico-Injector (Harvard Apparatus). Distinct morpholinos were designed that either target near the start codon to block mRNA translation (TCTTCCTCCGTCTCCCCCATCCTTC - (SEQ ID NO: 46)) or at the third intron-exon junction to block splicing
  • AGAAGTTGCTTCAGAACTCACTGAT (SEQ ID NO: 48)). Both morpholinos - one blocking exonl-intronl and one blocking exon2-intron2 gave the same results. Injection of 2 ng of the translation-blocking morpholino or 2-4 ng of this splice-blocking morpholino was sufficient to generate reproducibly cardiomyopathy, which for the splice-blocker correlates with loss of normal transcript. Embryos were anesthetized using Tricaine (United States Biochemical) prior to imaging. Brightfield and fluorescent images were taken using a Nikon SMZ1500 fluorescence microscope with an Insight Firewire 2 digital camera and SPOT advanced software.
  • embryos were fixed at the desired developmental stage and antibody stained to enhance the signal from the endogenous fluorescent transgene. Following overnight fixation in 4% paraformaldehyde (Sigma) at 4°C, embryos were rinsed four times for 20 minutes each in PBS-Tween (Fisher) (0.1% Tween in IX PBS), permeabilized for 25 minutes in cold acetone (Fisher), and then rinsed again four times for 20 minutes each in PBST. Embryos were blocked in 2% Bovine Serum Albumin (BSA) (Sigma) in PBST for 2 hr and then incubated with the primary antibody diluted in blocking reagent overnight at 4°C. Following four washes in
  • BSA Bovine Serum Albumin
  • PBST the embryos were incubated in secondary antibody diluted in blocking reagent for 2 hr at room temperature and then rinsed four times for 20 minutes each in PBST prior to imaging.
  • the following primary antibodies were used at a concentration of 1 :500: anti-green fluorescent protein mouse IgG2a, monoclonal 3E6 (Invitrogen) and MF20 polyclonal (Developmental Studies Hybridoma Bank).
  • the following secondary antibodies were used at a concentration of 1 :500: Alexa 488 goat anti mouse IgG2a (Invitrogen) and Alexa 568 goat anti rabbit IgG (Invitrogen).
  • RNAs were generated in vitro from linearized templates using the pCS2 expression vector and the mMessage mMachine kit (Invitrogen).
  • RNAs were generated in vitro from linearized templates using the pCS2 expression vector and the mMessage mMachine kit (Invitrogen).
  • 4 ng of RNA was injected, and >30 embryos were harvested and combined to generate RNA in each sample, using Trizol reagent as described above.
  • the sequence of qPCR primers is given in Table S2. Embryos were treated with CCG-1423 at 3 ⁇ in E3 buffer containing 1% DMSO.
  • Anti-MICAL-1, anti-MICAL-2 and anti-MICAL-3 antisera were produced in rabbits immunized with the KLH (keyhole limpet hemocyanin) protein conjugated with either CGGGWRAQLQPNPPA (SEQ ID NO: 49) (MICAL-1),
  • CGGRKNYGENAD SEQ ID NO: 50
  • MICAL-3 CGGKDYRSFHK
  • DRG neurons and adherent cell lines were rinsed with phosphate-buffered saline, pH 7.4 (PBS) and fixed with 4% paraformaldehyde (PFA) in cytoskeletal buffer (60 mM PIPES, 27 mM HEPES, 10 mM EGTA, 4mM MgS04 and 0.5% sucrose) for 30 min at room temperature.
  • PBS phosphate-buffered saline, pH 7.4
  • PFA paraformaldehyde
  • the cells were permeabilized for 20 min with PBS/0.5% Triton X-100, cross-fixed for 5 min in 4% PFA in PBS, washed twice with PBS/0.1% Triton-XlOO (PBST), blocked for 30 min in blocking buffer (PBST with 3% BSA) and labeled with primary antibodies in blocking buffer for 1 hr at room temperature or overnight at 4°C. Coverslips were washed three times with PBST and incubated with Alexa Fluor- conjugated goat secondary antibodies (Invitrogen) in blocking buffer for 1 h at room
  • coverslips were washed three times with PBS, once with water, and then mounted on a glass microscope slide (VWR) with Prolong Gold with DAPI (Invitrogen).
  • the following primary antibodies were used: rabbit anti-GFP (1 :2,000; Abeam, abl3970), goat anti-MRTF-A (1 : 100; SCBT C-19), lamin-A/C (1 : 100; Genscript A01455), and mouse anti- -actin (1 :500; Genscript, A00702).
  • Alexa Fluor-conjugated secondary antibodies were used at 1 : 1000.
  • F- and G-actin were visualized with Phalloidin 568 and DNAse I 594 respectively (Invitrogen). F-actin was stained by incubation with 0.3 ⁇
  • lentiviral constructs were prepared using a third-generation lentiviral system. Briefly, three helper plasmids (pLPl, pLP2, pVSV-G) and the vector containing the gene of interest with cis-acting sequences for proper packaging were used to generate pseudovirions. A subconfluent culture of HEK293T cells was transfected using the CalPhos Mammalian Transfection Kit (Clontech).
  • the pseudoviral particles were purified and concentrated from the supernatant collected 24 and 48 hr after transfection by ultracentrifugation at 22,000 rpm for 2 hr. Viral pellets were resuspended in PBS containing 1% BSA, aliquoted and stored at -80°C. The titer of each virus was determined using HEK293-T cells.
  • pseudoviral particles were added to the culture 1 hr after plating of the neurons, to allow cells to attach before transduction.
  • shRNAs were designed using iRNAi software
  • each shRNA was assessed by Western blotting of endogenous protein from dissociated DRG cells that had been infected with the viruses upon plating and were cultured for 5 days. The two shRNAs with the strongest knockdown efficiency were selected for further experiment so long as they knocked down greater than 75% of the protein. Cell health was assayed using the Dead End fluorometric tunel system (Promega)
  • Subcellular fractionation was performed using standard procedures (Cox and Emili, 2006). In brief, 24 hr after plating, an 80% confluent T 150 flask of HEK293T cells was scraped from the plate and lysed in 2 mL fractionation buffer (250 mM sucrose, 50 mM Tris-HCl pH 7.4, 5 mM MgC12, 1 mM DTT, and 1 tablet Roche Complete PIC). The lysate was then passed through a 25-gauge needle 10 times using a 5 mL syringe. The nuclei of the adherent cells were pelleted by centrifuging the lysates at 800 x g for 15 min.
  • 2 mL fractionation buffer 250 mM sucrose, 50 mM Tris-HCl pH 7.4, 5 mM MgC12, 1 mM DTT, and 1 tablet Roche Complete PIC.
  • the lysate was then passed through a 25-gauge needle 10 times using a 5 m
  • the nuclei were then washed twice with 2 mL fractionation buffer and spun down as previously described.
  • the nuclei were then resuspended in 300 lysis buffer, sonicated 3 x 30 sec and frozen at -80°C.
  • the supernatant that was set aside was then centrifuged at 6,000 x g to pellet the mitochondria.
  • the mitochondrial pellet was washed similarly to the nuclei pellet.
  • the supernatant from the mitochondrial pellet was then spun for 1 hr at 100,000 x g. The pellet from this spin was the microsomal fraction, while the supernatant was the pure cytosolic fraction.
  • MICAL proteins Purification of MICAL proteins.
  • a novel protein expression construct was generated in order to purify the redoxCH domains of MICAL- 1 (amino acids 1-611), -2 (amino acids 1-622) and -3 (amino acids 1-623) in sufficient quantities for enzymatic assays.
  • the expression construct used NusA to increase the solubility of the redoxCH domain of each MICAL, as described previously (Hung et al, 2010).
  • the protein-solubilizing factor NusA was cloned immediately after the His6-tag in pCold DNA I with a TEV protease cleavage site immediately 3 ' to the NusA protein.
  • TEV protease and 14 mM ⁇ -mercaptoethanol (BME) were added to the lysates under dialysis and the His-NusA protein was cleaved from MICAL at 4oC overnight. After 12 hr the lysates was dialyzed against lysis buffer to remove the ⁇ - mercaptoethanol and then bound to streptactin resin (IBA). The streptactin resin was washed with wash buffer (50 mM sodium phosphate pH 8.0 300 mM NaCl) and then the purified MICAL protein was eluted with 2.5 mM D-desthiobiotin. The D-desthiobiotin was removed on a desalting column (Biorad) and the protein was concentrated to 1 mg/mL on an Amicon spin column (Amicon 10k MWCO). The purified MICAL protein was analyzed for purity by
  • Polymerization was induced by the addition of lOx polymerization buffer (500 mM KC1, 20 mM MgC12, and 5 mM ATP) to a final concentration of lx.
  • MICAL- 1, -2, or -3 and/or NADPH was then added and the fluorescence intensity was immediately monitored every 15 sec over 20 min. Fluorescence intensity was measured on a SpectraMax M2e at 407 nm with an excitation wavelength of 365 nm.
  • NADPH depletion assays were performed similarly except with unlabeled actin instead of pyrene actin. The absorbance of NADPH was subsequently read at 365 nm.
  • qPCR Isolation of mRNA and qPCR was performed as follows. 50,000-100,000 cells were plated in 12-well plastic dishes. 24 hr later the RNA in the lysates was extracted using the Trizol protocol. The RNA was resuspended in 50 ⁇ H20 at a concentration of 1 ⁇ g/ ⁇ L. cDNA was transcribed using the Superscript III First-Strand Synthesis SuperMix for qRT-PCR using random hexamers in a 20 ⁇ reaction. cDNAs were amplified using Phusion PCR mastermix.
  • MICAL-2 is enriched in the nucleus. Because of the important roles of
  • MICAL-1 in depolymerizing actin in axonal growth cones we sought to understand if MICAL-2 and -3 have related functions.
  • MICAL-2 and -3 we examined the subcellular localization of each MICAL isoform. Cherry-tagged MICAL- 1 was localized to the cytoplasm in HEK293T cells, consistent with the role of MICAL-1 in F-actin disassembly in the cytoplasm. However, Cherry-MICAL-2 and Cherry-MICAL-3 were localized to the nucleus ( Figure 1A). To confirm that these localizations are not due to the presence of the Cherry tag, we monitored the localization of endogenous MICALs by subcellular fractionation.
  • MICAL-2 redox activity induces actin depolymerization. Because MICAL-2 and -3 share 60% and 63% identity, respectively, with MICAL-1 in the catalytic flavoprotein monooxygenase domain, we asked if they also depolymerize F-actin. To test this, we purified the bacterially expressed catalytic domain and the adjacent actin-binding calponin homology (CH) domain (designated 'redoxCH') for each MICAL isoform (Hung et al, 2010, Nature 463, 823-827).
  • CH actin-binding calponin homology
  • MICAL-2 regulates nuclear actin polymerization.
  • MICAL-2 and -3 to depolymerize F-actin in vitro, combined with their distinct nuclear localization in vivo, suggests that these isoforms may regulate actin polymerization within the nucleus.
  • F-actin in the nucleus is typically present as short, dynamic polymers that are difficult to detect using standard phalloidin staining. We therefore examined the effect of inhibiting MICAL-2 activity on F-actin levels within the nucleus in various cell lines.
  • MICAL-2 CT which contains a deletion of the N-terminal catalytic domain
  • MICAL-2GV which contains a glycine to valine mutation at amino acid 95 that blocks FAD-binding in flavoprotein monooxygenases
  • MICAL-2 expression induces nuclear localization of MRTF-A. Since
  • MICAL-2 regulates F-actin within the nucleus, and since nuclear G-actin mediates nuclear export of MRTF-A, we considered the possibility that MICAL-2 would reduce nuclear MRTF-A by promoting its export.
  • endogenous MRTF-A localization by immunofluorescence in HEK293T cells following MICAL-2 overexpression.
  • cells were infected with a virus expressing either GFP or GFP-MICAL-2 and serum-starved for 18 h under low-serum conditions (0.3% fetal bovine serum [FBS]) ( Figures 2A and 2B). Contrary to our expectation, overexpression of MICAL-2 under these conditions caused a marked increase in nuclear localization of MRTF-A ( Figures 2A and 2B).
  • MICAL-2 induces stress fiber formation. Stress fiber formation in the cytosol of NIH 3T3 cells has been shown to deplete nuclear actin, thereby leading to an accumulation of MRTF-A in the nucleus ( Figure 9H). However, MICAL-2 expression did not induce stress fibers or increase cytosolic F-actin levels in HEK293T cells ( Figures 9I-L). Taken together, these data indicate that MICAL-2 does not increase nuclear MRTF-A through increasing cytosolic F-actin or triggering stress fiber formation.
  • MICAL-2 mediates NGF-dependent nuclear localization of MRTF-A.
  • MICAL-2 mediates NGF-dependent nuclear localization of MRTF-A after growth factor stimulation.
  • NGF signaling is known to result in MRTF-A localization to the nucleus and activation of SRF/MRTF-A-dependent gene transcription.
  • MICAL-2 is required for NGF-mediated induction of
  • MRTF-A nuclear localization in response to NGF was markedly reduced by expression of either of two MICAL-2-specific shRNAs, but not by a control shRNA ( Figures 2C and 2D). These data indicate that MICAL-2 promotes nuclear localization of MRTF-A in response to NGF.
  • MICAL-2 expression induces the SRF/MRTF-A-dependent reporter.
  • the ability of MICAL-2 to induce nuclear localization of MRTF-A suggests that MICAL-2 is a regulator of SRF/MRTF-A-dependent gene transcription.
  • MRTF-A-ATAD a dominant-negative MRTF-A mutant. This protein lacks the transcription activation domain (TAD) that is necessary for an active SRF/MRTF-A complex. Expression of MRTF-A-ATAD blocked the effect of MICAL-2 on the SRF/MRTF-A reporter ( Figure 3D). These data indicate that MICAL-2 induces the reporter in an MRTF-A-dependent manner.
  • MICAL-2 expression induces endogenous SRF/MRTF-A target gene expression.
  • MRTF-A-independent SRF-dependent genes TSP1, PTGS1, and Egr2. These data indicate that MICAL-2 expression selectively activates SRF/MRTF-A-dependent gene expression.
  • MICAL-2 induces SRF/MRTF-A-dependent gene expression in PC12 cells.
  • Expression of MICAL-2 in PC12 cells similarly induced previously characterized SRF/MRTF-A target genes Acta2, Cyr61, and SRF by 100-200% ( Figure 1 1C).
  • MICAL-2 expression did not induce SRF-dependent, MRTF-A-independent genes, such as TSP1, PTGS1, and Egr2 ( Figure 11C). These data indicate that MICAL-2 expression induces endogenous SRF/MRTF-A-dependent gene expression.
  • MICAL-2 is required for serum and NGF-induced SRF/MRTF-A target gene expression.
  • FIG. 1 IB Conversely, MICAL-2-specific shRNA did not affect the induction of MRTF-A- independent SRF-dependent genes TSP1, PTGS1, and Egr2.
  • NGF induces the expression of Acta2, Cyr61, VCL, and SRF in an SRF- and MRTF-A-dependent manner ( Figure 1 ID).
  • MICAL-2 shRNA impaired NGF-induced expression of these SRF/MRTF-A target genes ( Figure 1 ID), but did not affect NGF-mediated induction of MRTF-A-independent SRF- dependent genes TSP1 and PTGS1.
  • rat El 4- 15 DRG neurons cultured in NGF exhibited a reduction in Acta2, Cyr61 and VCL expression upon infection with lentivirus expressing either of two MICAL-2 shRNAs, while MRTF-A-independent SRF-dependent genes TSP1, PTGS1 and Egr2 were unchanged ( Figure 1 IE).
  • MICAL-2 is required for mediating serum- and NGF-dependent increases in SRF/MRTF-A- dependent gene expression.
  • MICAL-2 is required for NGF-dependent neurite outgrowth.
  • MICAL-2 mediates NGF-induced SRF/MRTF-A-dependent transcription
  • PC 12 cells and embryonic DRG sensory neurons we monitored the effect of MICAL-2 knockdown on NGF-dependent outgrowth in PC 12 cells and embryonic DRG sensory neurons. Neurite outgrowth in these cells is considered to reflect activation of SRF/MRTF-A-dependent gene transcription.
  • MICAL-2-specific shRNA neurite length 4 d after NGF treatment was reduced by -40% compared to LacZ shRNA-infected PC 12 cells ( Figures 4A and 4B).
  • Knockdown of MICAL-2 in differentiated PC12 cells also decreases neurite formation after NGF treatment ( Figure 4C).
  • Embryonic MICAL-2 knockdown impairs SRF/MRTF-A-dependent gene expression.
  • MICAL-2 is required for SRF/MRTF-A-dependent gene transcription induced by physiologic signals.
  • SRF-dependent gene transcription is required for heart development in zebrafish.
  • MICAL isoforms have been characterized in zebrafish, with the cardiac-enriched micaUb exhibiting highest sequence identity to mouse MICAL-2. Therefore, zebrafish cardiac development provides a useful system to explore whether MICAL-2 promotes SRF/MRTF-A-dependent gene expression elicited by physiologic signals.
  • phenotypes include small hearts that failed to induce normal looping at 24 hpf ( Figures 5A and 5B), and thin, linear morphology compared to wild-type hearts at 48 hpf ( Figures 5C and 5D). Additionally, cardiomyocytes in morphant hearts were spatially disorganized, rather than displaying the relatively even distribution seen in wild-type hearts, as shown at 52 hpf in the atria ( Figures 5E-G). Taken together, these data confirm that mical2b expression was effectively knocked down in zebrafish.
  • MICAL-2 does not activate SRF/MRTF-A through RhoA.
  • RhoA inhibition affects MICAL-2 -dependent induction of the SRF/MRTF-A reporter.
  • application of cell-permeable C3 -transferase, an inhibitor of RhoA, or Y-27632, a ROCK inhibitor did not block MICAL-2-induced activation of the SRF/MRTF-A reporter in serum-starved HEK293T cells ( Figure 6A).
  • MICAL-2 induces depletion of nuclear actin.
  • MICAL-2 activates SRF/MRTF-A-dependent gene transcription by depleting nuclear G-actin.
  • MICAL-2 might also reduce nuclear G-actin levels.
  • MICAL-2 affects nuclear G-actin levels
  • HEK293T cells were infected with MICAL-2-expressing lentivirus and subsequently serum-starved.
  • MICAL-2 overexpression caused a significant reduction in nuclear G-actin even in the absence of serum ( Figures 6C and 6D).
  • the reduction in nuclear G-actin induced by MICAL-2 was similar to the reduction induced by serum stimulation ( Figures 6C and 6D).
  • the SRF/MRTF-A pathway inhibitor CCG-1423 is a MICAL-2 inhibitor.
  • CCG-1423 is a small molecule inhibitor of SRF/MRTF-A-dependent transcription.
  • CCG-1423 has promising effects in conditions associated with increased SRF/MRTF-A signaling, such as metastasis of melanoma cells in vitro and in vivo, and insulin resistance in animals. While CCG-1423 has shown utility in various preclinical disease models, its specific molecular target is not known.
  • T m melting temperature
  • 1,8-ANS l-anilinonaphthalene-8-sulfonic acid
  • MICAL-2 activity we used an NADPH depletion assay. Recombinant MICAL-2, comprising the enzymatic domain and the CH domain (MICAL-2 redoxCH ), was incubated with NADPH in the presence of F-actin, and the consumption of NADPH was monitored by UV absorbance.
  • MICAL-2 SRF/MRTF-A-dependent gene expression. Additionally, knockdown of MICAL-2 markedly reduces NGF-stimulated SRF/MRTF-A-dependent expression and neurite growth.
  • Our findings identify MICAL-2 as a novel regulator of SRF/MRTF-A signaling that acts by regulating nuclear actin. We also found that that physiologic regulation of SRF/MRTF-A-dependent gene transcription in vivo is impaired by knockdown of the zebrafish MICAL-2 ortholog. These studies identify MICAL-2 as a physiologic regulator of SRF/MRTF-A signaling in diverse cell types.
  • MICAL-2 -mediated increase in nuclear MRTF-A levels appears to be caused by a reduction in nuclear G-actin levels.
  • MRTF-A loses the cofactor needed for its export, resulting in nuclear accumulation.
  • the ability of MICAL-2 to depolymerize nuclear F-actin originally suggested to us that MICAL-2 would increase nuclear G-actin levels, and therefore decrease MRTF-A levels in the nucleus.
  • MICAL-2 expression lead to increased nuclear levels of MRTF-A.
  • MICAL-2 expression reduced overall nuclear G-actin levels, to the same degree that is seen following serum and NGF stimulation.
  • MICAL isoforms constitute a family of actin regulatory proteins that influence actin polymerization through a redox modification, each protein appears to exhibit distinct localizations and functions. While MICAL-1 is cytosolic, MICAL-2 and -3 appear to be enriched in the nucleus.
  • MICAL-2 The effects of MICAL-2 that we see in HEK293T cells, PC 12 cells, and DRG neurons are unlikely to reflect cytosolic functions of MICAL-2 since MICAL-2 is predominantly nuclear in these cell types, and because the effects of MICAL-2 on SRF/MRTF- A-dependent gene transcription are not seen following expression of MICAL-2 constructs that contain mutations in the nuclear-localization sequence. Thus, the effects of MICAL-2 reflect its ability to depolymerize nuclear F-actin.
  • This example illustrates the use of the methods described herein to screen potential candidate agents that modulate MICAL-2 activated SRF/MRTF-A pathway.
  • the pyrene-actin assay to determine conversion of F-actin to G-actin was used as an indication of the effect on MICAL-2 activity. Based on this assay, aurin tricarboxylic acid was identified.
  • aurin tricarboxylic acid potently prevented MICAL-2 from depolymerizing pyrene-labeled F-actin at all concentrations tested (20 ⁇ , 10 ⁇ , 5 ⁇ , 2.5 ⁇ , 1.25 ⁇ , 0.625 ⁇ , 0.3125 ⁇ , 0.156 ⁇ , 0.078 ⁇ , 0.039 ⁇ ) indicating a IC50 value of below 40 nM.
  • Aurin tricarboxylic acid was counter screened to demonstrate that it was not fluorescent itself and that it did not increase the level of F-actin polymerization in the well.
  • This example describes another target in the regulation of SRF/MRTF-A pathway.
  • the general materials and methods used were the same as in Example 1.
  • neither of these genes contain SRF/MRTF-A response elements and neither is known as a SRF/MRTF-A target gene.
  • MICAL-2 does not regulate Axl and Gas6 expression, or vice-versa.
  • MICAL-2 to mediate signaling downstream of Gas6 and Axl. If MICAL-2 is downstream of Axl, then activation of Axl should induce SRF/MRTF-A-dependent gene expression.
  • Axl was transfected into HEK293 cells cultured in media containing minimal serum (0.3% FBS). In GFP-transfected cells, minimal levels of luciferase were detected (Fig. 15a). However, expression of GFP-Axl resulted in an approximately 4 fold increase in luciferase expression (Fig. 15a). Further, we found that Addition of Gas6 to HEK293 cells expressing the
  • MRTF-A localization in GFP-Axl expressing cells HEK293 cells were transfected with GFP or GFP-Axl and endogenous MRTF-A localization was measured by immunofluorescence. In these experiments, cells were cultured in minimal serum (0.3% FBS). In GFP-expressing cells, minimal nuclear localization of MRTF-A was detected. However, in GFP-Axl expressing cells, nuclear localization of MRTF-A was readily detected (Fig. 15d,e). In adjacent non-transfected cells, MRTF-A remained in the cytosol.
  • screening for interference with MICAL-2 activity can also identify agents that impair the actions of Gas6/Axl signaling pathways. Such agents may be useful for the treatment of diseases that involve elevated Axl signaling, such as cancer and metastatic disease.

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Abstract

L'invention concerne des procédés pour l'identification d'agents qui interfèrent avec l'activation de la voie SRF/MRTF induite par MICAL-2. Le procédé consiste à évaluer l'activité des agents candidats sur les différents points dans la voie.
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EP3733837A4 (fr) * 2017-12-28 2021-10-06 Kaneka Corporation Agent favorisant l'agrégation de cellules
CN110331156A (zh) * 2019-05-30 2019-10-15 深圳大学 抗Mical2多克隆抗体及其制备方法
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