Stabilization of Я-Catenin by a Wnt-Independent Mechanism

Stabilization of ␤-catenin by a Wnt-independent mechanism regulates cardiomyocyte growth

Syed Haq*†, Ashour Michael*, Michele Andreucci‡, Kausik Bhattacharya*, Paolo Dotto‡, Brian Walters*, James Woodgett§, Heiko Kilter*, and Thomas Force*†

*Molecular Cardiology Research Institute, Tufts–New England Medical Center, and Tufts University School of Medicine, Boston, MA 02111; ‡Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; and §University of Toronto, Toronto, Ontario, Canada M5S 1A1

Communicated by Alexander Leaf, Harvard University, Charlestown, MA, February 21, 2003 (received for review September 30, 2002) ␤-Catenin is a transcriptional activator that regulates embryonic phorylation inhibits GSK-3 activity directed toward primed development as part of the Wnt pathway and also plays a role in substrates that have been previously phosphorylated at a site tumorigenesis. The mechanisms leading to Wnt-induced stabiliza- four residues carboxy terminal to the GSK-3 phosphorylation tion of ␤-catenin, which results in its translocation to the nucleus site but does not inhibit kinase activity directed toward unprimed and activation of transcription, have been an area of intense substrates (15, 16). This mechanism is used in growth factor interest. However, it is not clear whether stimuli other than Wnts signaling but is not believed to be important in Wnt signaling and can lead to important stabilization of ␤-catenin and, if so, what has been reported to be insufficient to induce ␤-catenin accu- factors mediate that stabilization and what biologic processes mulation (17, 18). Although these data are compatible with might be regulated. Herein we report that ␤-catenin is stabilized in ␤-catenin being unprimed in situ (19), recent studies indicate that cardiomyocytes after these cells have been exposed to hypertro- ␤-catenin can exist as a primed target for GSK-3, when phos- phic stimuli in culture or in vivo. The mechanism by which ␤-catenin phorylated on Ser-45 by casein kinase 1␣ (20), and raise the is stabilized is distinctly different from that used by Wnt signaling. possibility that, in certain circumstances, Ser-9 phosphorylation Although, as with Wnt signaling, inhibition of glycogen synthase of GSK-3␤ could stabilize ␤-catenin. A second mechanism of kinase-3 remains central to hypertrophic stimulus-induced stabili- inhibition of GSK-3, used by Wnts, involves, in part, complex zation of ␤-catenin, the mechanism by which this occurs involves formation of GSK-3 with GSK-3-binding protein͞Frat1 (21, 22). the recruitment of activated PKB to the ␤-catenin-degradation Complex formation is believed to sequester GSK-3 and primarily complex. PKB stabilizes the complex and phosphorylates glycogen inhibit phosphorylation of unprimed substrates, at least in kinase synthase kinase-3 within the complex, inhibiting its activity di- assays in vitro (12, 15). rected at ␤-catenin. Finally, we demonstrate via adenoviral gene Increases in ␤-catenin levels in the cytosol, together with less transfer that ␤-catenin is both sufﬁcient to induce growth in well defined signals, lead to its translocation to the nucleus, cardiomyocytes in culture and in vivo and necessary for hypertro- where it acts in tandem with T cell factor (Tcf)͞lymphocyte phic stimulus-induced growth. Thus, in these terminally differen- enhancer factor (Lef) family members to induce expression of ␤ tiated cells, -catenin is stabilized by hypertrophic stimuli acting several genes involved in cell cycle reentry, as well as in transfor- via heterotrimeric G protein-coupled receptors. The stabilization mation of postnatal cells (23, 24). In this manuscript, we ask what occurs via a unique Wnt-independent mechanism and results in role, if any, this pathway might be playing in terminally differenti- cellular growth. ated cells that cannot enter the cell cycle and whether the mechanisms regulating ␤-catenin stability differed in these cells (11). lycogen synthase kinase-3 (GSK-3)-␣ and -␤ function as Ginhibitors of Wnt signaling during the development of the Methods embryonic axis (1). GSK-3 is also a negative regulator of growth Adenoviruses. AdGFP, Ad␤-catenin, and Ad␤-catenin⌬ contain in cardiomyocytes, cells that are terminally differentiated and cytomegalovirus-driven expression cassettes for enhanced GFP can only undergo hypertrophic growth (2–4). Inhibition of and either ␤-galactosidase or vesicular stomatitis virus-tagged GSK-3 is necessary for the hypertrophic response both in vitro ␤-catenin or ␤-catenin⌬ (␤-catenin deleted for the N-terminal and in vivo (2–5), and at least some of the antihypertrophic 134 amino acids, a region that contains the GSK-3 phosphory- effects of active GSK-3 are mediated by regulating activity of the lation sites), respectively, substituted for E1 through homologous nuclear factor of activated T cells (NF-AT) family of transcrip- recombination (24). AdGSK-3␤(S9A), encoding GSK-3␤ with a tion factors (2, 5, 6). However, we found that gene transfer of an Ser-9-to-Ala mutation has been described (2). AdNF-AT⌬, activated NF-AT3 failed to recapitulate the full hypertrophic provided by Jeffery Molkentin (Children’s Hospital Medical response and asked whether additional GSK-3 targets could play Center, Cincinnati), encodes NF-AT3 deleted for the first 317 a role. ␤-Catenin, which plays critical roles in development and amino acids and is constitutively active (6). tumorigenesis (7, 8), is one potential target. The protein exists in the cell in two pools, membrane associated and cytosolic. In Cell Culture. Neonatal rat ventricular myocytes (NRVM). Cardiomyo- the membrane, ␤-catenin links cadherins to the cytoskeleton (9). cytes were prepared from 1- to 2-d-old rats by using standard ␤-Catenin also functions as a transcriptional coactivator (8), the methods (2). source of this being the cytosolic pool, which is negatively S2-Wingless (Wg)-secreting cells. S2 cells expressing Drosophila Wg regulated by GSK-3. GSK-3 phosphorylates the amino-terminal under the control of the metallothionein promoter were as ␤ region of -catenin, targeting it for ubiquitination and degra- described (25). Production of Wg was induced by addition of dation by the proteasome (10, 11). ␤-Catenin is phosphorylated by GSK-3 when part of a complex that includes the scaffolding protein Axin and the adenomatous polyposis coli gene product, Abbreviations: GSK-3, glycogen synthase kinase-3; NF-AT, nuclear factor of activated T cells; APC (12). Inhibition of GSK-3 is therefore essential for the Tcf, T cell factor; Lef, lymphocyte enhancer factor; NRVM, neonatal rat ventricular ␤ myocytes; Wg, Wingless; CSA, cross-sectional area; TAC, thoracic aortic constriction; PE, stabilization and accumulation of -catenin. phenylephrine. GSK-3 activity is inhibited via two primary mechanisms. One, †To whom correspondence should be addressed at: Tufts–New England Medical Center, 750 phosphorylation of an amino-terminal serine residue (Ser-21 for Washington Street, Box 8486, Boston, MA 02111. E-mail: [email protected] or ␣, Ser-9 for ␤; ref. 13), is catalyzed by PKB (14). This phos- [email protected].

4610–4615 ͉ PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0835895100 Downloaded by guest on September 30, 2021 CdCl2 to the culture medium to a final concentration of 0.1 mM. Media was collected 6 h later. Although Drosophila in origin, when added to mammalian cells, Wg activates the Wnt pathway (see Fig. 1D).

Luciferase Assays. Plasmids: pTOPFlash contains three copies of the Tcf͞Lef binding site upstream of the thymidine kinase minimal promoter and luciferase cDNA. pFOPFlash has three copies of a mutated Tcf͞Lef binding site (Upstate Biotechnol- ogy). NRVMs were transfected by using FuGENE 6 (Roche) with 0.2 ␮g of plasmid per 1 ϫ 105 cells. Luciferase activity was determined by using a commercially available assay system (Promega) and was normalized for transfection efficiency with pcDNA3-␤-galactosidase.

[3H]Leucine Incorporation. Cardiomyocytes in 12-well dishes were infected with Ad␤-catenin, Ad␤-catenin⌬, AdNF-AT⌬,or AdGFP for 48 h. For the final 14 h, [3H]leucine (1 ␮Ci per well) was added. [3H]Leucine incorporation was determined as described (2).

Immunoprecipitation and Immunoblotting. Immunoblotting of whole-cell lysates from cardiomyocytes in culture or the intact heart was performed as described (2, 26). Lysates were routinely blotted with antibodies against actin or GAPDH to confirm equivalent loading. All immunoblots shown are representative of at least three.

Cytosolic Fractionation. NRVMs were fractionated by hypotonic lysis as described (2), except that the centrifugation was performed at 100,000 ϫ g. Immunoblotting with an anticaveolin antibody has confirmed that, with this protocol, there is no contamination of the cytosolic fractions with membrane components. Fig. 1. Hypertrophic stimuli induce ␤-catenin accumulation in vitro and in Immunocytochemistry. NRVMs on coverslips were transduced vivo.(A) NRVMs were treated with PE (100 ␮M), endothelin-1 (100 nM), or, as with viruses or transfected with plasmids, and cells were stained a positive control, with the GSK-3 inhibitor LiCl (10 mM), for the times indicated. Cytosolic fractions were blotted with anti-␤-catenin antibody. An- with antibodies noted in figure legends. Cardiomyocyte area was tiactin blot conﬁrms equivalent protein loading. (B) NRVMs were transfected determined as described (2) by using OPENLAB software from with pTOPFlash or pFOPFlash and 24 h later were stimulated with PE or vehicle Improvision, Inc. (Quincy, MA). (Cont) for an additional 24 h before luciferase assay. Luciferase activity of vehicle-treated, pTOPFlash-transfected cells was set at 1, and other results are Adenovirus Injections in Vivo. Rats were anesthetized, and the expressed as fold activation relative to that value (n ϭ 5 independent exper- myocardium of the left ventricle was directly injected at five sites iments, done in triplicate; *, P Ͻ 0.01 vs. all other values). (C) Rats were on the lateral free wall with 1 ␮l(1ϫ 1011 particles per milliliter) subjected to TAC or sham surgery (control) for the times indicated. Lysates of of AdGFP, or Ad␤-catenin⌬ diluted in 10 ␮l of PBS. Eight days cytosolic fractions were blotted with anti-␤-catenin antibody. Anti-GAPDH ␤ after injection, the hearts were arrested in cardioplegia solution blot conﬁrms equivalent loading. Shams had no increase in -catenin through- out the 7-d protocol (data not shown). (D Top), NRVMs and HEK293 cells were and fixed. GFP-positive cardiomyocytes were identified in trans- treated for 2 h with media from S2 cells that had not been induced to secrete verse sections by using an anti-GFP antibody (Santa Cruz Wg (NI) or were induced (I) by the addition of CdCl2 (see Methods). A separate Biotechnology), followed by a horseradish peroxidase- group of cells was pretreated with Frizzled-related protein-1 (FRP, 15 ␮M) for conjugated secondary Ab, and counterstained with hematoxylin. 1 h before the addition of NI or I S2 media for 2 h. Cytosolic fractions were Cardiomyocyte cross-sectional area (CSA) was determined on blotted with anti-␤-catenin antibody. (Middle) NRVMs were incubated with GFP-positive myocytes (infected with AdGFP or Ad␤-catenin⌬) PE (100 ␮M), with or without Frizzled-related protein-1 pretreatment for 1 h, and adjacent nontransduced myocytes. for the times indicated. Cytosolic fractions were blotted with anti-␤-catenin antibody. (Bottom) NRVMs were treated with or without PE for 14 h, after Thoracic Aortic Constriction (TAC). Rats were subjected to supra- which the conditioned media were collected and incubated for the times indicated with fresh cultures of NRVMs. Cytosolic fractions were blotted with valvular aortic banding as described (26). Control rats underwent anti-␤-catenin antibody. a sham operation.

Results CELL BIOLOGY ␤ reporter, pTOPFlash, but not from a mutated Tcf͞Lef reporter, Stabilization of -Catenin by Hypertrophic Stimuli. We examined the ␤ response of NRVMs to hypertrophic agonists. Phenylephrine pFOPFlash (Fig. 1B). -Catenin was also stabilized in vivo in the (PE) and endothelin-1 induced a biphasic pattern of stabilization rat heart exposed to the hypertrophic stress of pressure overload, of ␤-catenin with an initial peak at 90–120 min, a return to although, unlike the cardiomyocytes in culture, there was no ␤ baseline by 3 h, and then a much more sustained peak lasting early peak after TAC (Fig. 1C). -Catenin levels increased at 1 d from Ϸ6 to 24 h (Fig. 1A). The increase in cytosolic ␤-catenin and peaked by3dafterTAC(Fig. 1C). was equivalent to that induced by direct inhibition of GSK-3␤ One mechanism by which hypertrophic agonists could stabi- activity with LiCl (Fig. 1A). Importantly, this accumulation of lize ␤-catenin is by inducing release of a Wnt from cardiomyo- ␤-catenin was sufficient to activate transcription from a Tcf͞Lef cytes and recruiting the classical Wnt pathway. We found that

Haq et al. PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4611 Downloaded by guest on September 30, 2021 Fig. 2. Mechanisms of ␤-catenin stabilization in cardiomyocytes. NRVMs were stimulated with PE (100 ␮M) for the times indicated. (A) Lysates were subjected to immunoprecipitation with anti-Frat1 antibody followed by blotting with antibodies to GSK-3␤ or Frat1. (B) Lysates were blotted with antibodies to Ser-9-phosphorylated GSK-3␤ (p-GSK-3␤) or to GSK-3␤.(C) Lysates were subjected to immunoprecipitation with anti-Axin antibody followed by blotting with antibodies to p-GSK-3␤, GSK-3␤, Axin, or with an antibody recognizing Ser-473-phosphorylated (active) PKB (p-PKB) or total PKB. (D) NRVMs were pretreated with either DMSO or lactacystin (10 ␮M) for 1 h before stimulation with PE for the times indicated. Cytosolic fractions were prepared and then blotted with anti-phospho-speciﬁc Ser-33͞37͞Thr-41 ␤-catenin antibody. (E and F) NRVMs were transduced with AdPKB-AA, AdPKB-myr, or AdGFP (control) for 48 h by using a multiplicity of infection of 25 plaque-forming units per cell and then stimulated with PE for the times indicated. Cell lysates were blotted with antibodies to PKB, p-GSK-3␤, and GSK-3␤ (E), or lysates were immunoprecipitated with anti-Axin antibody followed by blotting with antibodies to PKB, p-GSK-3␤, GSK-3␤ (F). Cytosolic fractions were blotted with anti-␤-catenin antibody (Bottom). (G) NRVMs were transduced with AdPKB-AA and 48 h later were incubated for 2 h with LiCl, lactacystin, or DMSO, as indicated. Cytosolic fractions were blotted with anti-␤-catenin antibody. (H) NRVMs were transduced with either AdGSK-3␤(S9A) or AdGFP for 24 h and then stimulated with PE or vehicle (-) for 14 h. Cytosolic fractions were blotted with anti-␤-catenin antibody.

stimulation of cardiomyocytes with the Drosophila Wnt ho- of GSK-3 is believed to be that which is complexed with Axin in the molog, Wg, led to stabilization of ␤-catenin, although the ␤-catenin-degradation complex and, therefore, has access to response was relatively weak compared with that of HEK293 ␤-catenin. The amount of GSK-3␤ in the complex that was phos- cells (Fig. 1D Top). Stabilization of ␤-catenin in response to Wg phorylated on Ser-9 increased markedly, coincident with both the was blocked in both HEK293 cells and cardiomyocytes by early peak (data not shown) and the late peak of PE-induced addition to the media of Frizzled-related protein-1 (provided by ␤-catenin accumulation (Fig. 2C). Thus, although GSK-3␤ was Jeffrey Rubin, National Cancer Institute, Bethesda), which binds present in the Axin complex, and therefore had access to ␤-catenin, to Wg and Wnts, blocking activation of the Wnt pathway (Fig. the GSK-3␤ was phosphorylated on Ser-9 and inhibited. 1D Top and ref. 27). In contrast, stabilization of ␤-catenin by PE We also examined a time course of phosphorylation of was not blocked by Frizzled-related protein-1, suggesting it was ␤-catenin on three GSK-3␤ sites, Ser-33, Ser-37, and Thr-41, not mediated by recruitment of the Wnt pathway (Fig. 1D reasoning that this should correlate with the activation state of Middle). In addition, PE-conditioned media did not lead to the kinase and inversely correlate with Ser-9 phosphorylation, if stabilization of ␤-catenin (Fig. 1D Bottom). These data suggest phosphorylation of GSK-3␤ on Ser-9 is the critical determinant that PE-induced stabilization of ␤-catenin is not mediated by of stabilization of ␤-catenin. We were unable to detect any recruitment of the classical Wnt pathway and is also not sec- phosphorylation of ␤-catenin on these sites at any point in the ondary to release of a stable factor acting in a paracrine fashion. time course (Fig. 2D). However, this finding could be consistent We examined the mechanisms by which hypertrophic agonists with rapid clearance of ␤-catenin by the proteasome once these stabilized ␤-catenin in cardiomyocytes. We focused on the sites were phosphorylated. Therefore, we performed a PE time delayed͞sustained peak of ␤-catenin because, based on the course in the presence of the proteasome inhibitor lactacystin. absence of an early peak in vivo (Fig. 1C), the later peak We found significant phosphorylation of ␤-catenin only in the appeared to be more relevant to the hypertrophic response. We presence of lactacystin and only in the control and 5-h-after-PE found that sequestration of GSK-3␤ by Frat1 played no signif- time points, when GSK-3␤ was most active (i.e., not phosphor- icant role in inhibition of GSK-3␤, because the amount of ylated on Ser-9; Fig. 2D). Thus, at the control and 5-h time GSK-3␤ complexed with Frat1 decreased 6–14 h after PE (Fig. points, when GSK-3␤ is not phosphorylated on Ser-9 and is 2A), times at which ␤-catenin levels were increasing (Fig. 1A). active, inhibition of the proteasome exposes the phosphorylation We therefore turned to the alternative mechanism of GSK-3␤ of ␤-catenin on the GSK-3␤ sites. Conversely, when GSK-3␤ is inhibition, Ser-9 phosphorylation, and found an increase in phosphorylated on Ser-9 and inhibited (1.5 and 14 h after PE), phosphorylation at 3 h, which peaked at 14–24 h after PE (Fig. there is no detectable phosphorylation of ␤-catenin, even in the 2B), when ␤-catenin levels were maximal (Fig. 1A). Thus, the presence of lactacystin. These studies suggest a central role time course was consistent with Ser-9 phosphorylation playing a for Ser-9 phosphorylation of GSK-3␤ in the stabilization of role in the accumulation of ␤-catenin. However, the critical pool ␤-catenin by hypertrophic stimuli.

4612 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0835895100 Haq et al. Downloaded by guest on September 30, 2021 Coincident with the increase in phosphorylation of GSK-3␤ in the complex, active PKB, as evidenced by Ser-473 phosphorylation, was identified in the complex (Fig. 2C). To confirm that the association of activated PKB with the complex resulted in the observed phosphorylation of GSK-3␤, we expressed a dominant inhibitory mutant of PKB, PKB-AA, in which the activating phosphorylation sites, Ser-473 and Thr-308, were replaced with Ala residues (28). PKB-AA inhibited the PE-induced phosphorylation of total cellular GSK-3␤ (Fig. 2E Middle) and, as shown in Fig. 2F Middle, inhibited phosphorylation of Axin-associated GSK-3␤ on Ser-9, promoted destabilization of the Axin complex as evidenced by significant dissociation of GSK-3␤ from Axin, and blocked the cytosolic accumulation of ␤-catenin. Two alternative dominant negatives, PKB-K179M and PKB-AAA, produced comparable results (data not shown). Conversely, when we transduced cells with myristoylated and active PKB (28), we observed increased Ser-9 phosphorylation of total cellular GSK-3␤ (Fig. 2E) and Axin-associated GSK-3␤ (Fig. 2F Right), enhanced stabilization of the ␤-catenin-degradation complex, and accumulation of ␤-catenin (Fig. 2F Right). ␤-Catenin is most efficiently phosphorylated by GSK-3␤ and targeted for ubiquitination when it is in the Axin complex. Thus, the findings shown in Fig. 2F, demonstrating that expression of PKB-AA destabilizes ␤-catenin despite the fact that it disrupts the Axin complex, appear at odds with this principle. Therefore, we asked whether PKB-AA-induced destabilization of ␤-catenin might Fig. 3. Mechanisms of hypertrophic stress-induced stabilization of ␤-catenin beviaanAxin͞GSK-3␤-independent mechanism. We exposed in vivo. Rats were subjected to TAC for the times indicated. (A) Myocardial NRVMs that had been transduced with AdPKB-AA to either LiCl lysates were immunoprecipitated with anti-Frat1 antibody, followed by blot- ␤ or lactacystin. Both agents blocked PKB-AA-mediated degradation ting with anti-GSK-3 and anti-Frat1 antibodies. (B) Myocardial lysates were blotted with antibodies to p-PKB, PKB, p-GSK-3␤, or GSK-3␤.(C and D) Myo- of ␤-catenin (Fig. 2G). These data confirm that PKB-AA is acting ␤ cardial lysates were immunoprecipitated with an anti-Axin antibody followed predominantly via GSK-3 and the proteasome to destabilize ␤ ␤ ␤ ␤ ␤ by blotting with antibodies to GSK-3 , p-GSK-3 , and -catenin (C) or with -catenin, although a minor GSK-3 -independent mechanism can- anti-p-PKB or anti-PKB (D). not be ruled out. The data in Fig. 2 E–G suggest that, in the presence of PKB-AA, GSK-3␤ is maximally activated and efficiently phosphorylates ␤-catenin. Thus, even the minimal amount of Axin tion of ␤-catenin. However, at 1 and3dafterTAC, coincident complex evident in Fig. 2F Middle, appears to be sufficient to with the increase in ␤-catenin levels, there was a marked increase maintain ␤-catenin at low levels. Finally, we transduced cells with adenovirus encoding GSK- in the association of activated PKB with the complex (Fig. 3D) ␤ and a corresponding increase in phosphorylation on Ser-9 of 3 (S9A), a mutant unable to be inhibited by Ser-9 phosphorylation. ␤ This construct, which abrogates the hypertrophic response in car- GSK-3 (Fig. 3C). Thus, the mechanisms of stabilization of ␤ diomyocytes in culture and in vivo (2, 5), dramatically reduced the -catenin in vivo by hypertrophic stress appear to be very similar accumulation of ␤-catenin after PE (Fig. 2H). to those in cultured cardiomyocytes. In summary, the studies in NRVMs suggest that levels of ␤-catenin increase following hypertrophic stress, due, in large ␤-Catenin Regulates Growth of Cardiomyocytes. We next examined part, to GSK-3␤ phosphorylation on Ser-9, likely catalyzed by whether stabilization of ␤-catenin produced a phenotype in the recruitment of PKB to the Axin complex. Activated PKB also NRVMs. We transduced NRVMs with adenoviruses encoding enhances the stability of the complex. Furthermore, the accu- either vesicular stomatitis virus-tagged wild-type ␤-catenin or mulation of ␤-catenin is sufficient to induce transcription from ␤-catenin⌬, a stabilized mutant lacking the GSK-3 phosphory- promoters containing Tcf͞Lef elements. lation sites. Expression of the transgenes was confirmed (Fig. 4A Upper). Gene transfer with wild-type ␤-catenin led to only Mechanisms of Stabilization of ␤-Catenin in Vivo. We found that the modest increases in levels of ␤-catenin, indicating efficient ␤ mechanisms regulating stabilization of -catenin in the heart of compensation by the cells (Fig. 4A Upper). Gene transfer of the the intact rat exposed to pressure overload induced by TAC were stabilized mutant led to somewhat greater increases, although, similar to those in the PE-stimulated NRVMs. TAC induced the ␤ compared with endogenous levels, expression was increased only rapid dissociation of GSK-3 from Frat1 (Fig. 3A), suggesting Ϸ2- to 3-fold (Fig. 4A Upper). that sequestration of GSK-3 by Frat1 did not account for the The essential feature of hypertrophic growth, protein synthesis stabilization of ␤-catenin at 1 and3dafterTAC. (as determined by leucine incorporation), was significantly in- Phosphorylation of total cellular GSK-3␤ on Ser-9 increased creased by gene transfer of ␤-catenin⌬ (Fig. 4B). The increase after TAC, and the time course of the phosphorylation, which ⌬ CELL BIOLOGY peaked3dafterTAC, was compatible with this phosphorylation was equivalent to that induced by gene transfer of NF-AT ,a playing a role in stabilizing ␤-catenin (Fig. 3B). Phosphorylation of positive regulator of hypertrophy (6), and to endothelin-1 and total cellular PKB followed a similar profile of activation (Fig. 3B). only marginally less than that with PE (Fig. 4B). These changes We again examined signaling within the Axin complex. As were not attributable to proliferation of cardiomyocytes because ␤ ⌬ with the cells in culture, only more strikingly, complex formation -catenin did not induce cell cycle entry, as determined by of GSK-3␤ with Axin increased markedly after TAC, peaking at BrdUrd incorporation (data not shown). Another marker of 1–3 d (Fig. 3C). Furthermore, more ␤-catenin was present in the hypertrophy, cardiomyocyte size, was also significantly increased Axin complex after TAC (Fig. 3C), which would be expected to by ␤-catenin⌬ (Fig. 4C Left). Gene transfer of wild-type ␤- increase access of GSK-3␤ to ␤-catenin, leading to destabiliza- catenin increased protein synthesis and cardiomyocyte size

Haq et al. PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4613 Downloaded by guest on September 30, 2021 slightly but not significantly, compatible with the minimal increase in ␤-catenin levels achieved (Fig. 4 B and C Left). The prohypertrophic effects of ␤-catenin⌬ could have been mediated via effects at the cell membrane. However, coexpres- sion of a dominant inhibitory mutant of its transcriptional coactivator Lef-1, in which the DNA binding domain is pre- served but the N-terminal 56 amino acids, containing the ␤-catenin interaction domain, has been deleted (Lef-1⌬␤-cat; ref. 29), abrogated the ␤-catenin-induced increase in cell size (Fig. 4C Right). Of note, two other markers of hypertrophy in cardiomyocytes, sarcomere organization and expression of the fetal gene atrial natriuretic factor, were not induced by ␤-catenin (data not shown). These data confirm that ␤-catenin is sufficient to induce two of the critical components of the hypertrophic response, protein synthesis and cellular growth, and confirm that the effect is mediated via its transcriptional activating activity. We next asked whether ␤-catenin is necessary for the hypertrophic response of cardiomyocytes to physiological stimuli. ␤-Catenin constructs lacking the transcriptional activation domain do not consistently function as dominant inhibitory mutants because they can increase cytosolic and nuclear levels of ␤-catenin, possibly by displacing endogenous wild-type ␤-catenin from the membrane (30). Therefore, we again expressed Lef-1⌬␤-cat or wild-type Lef-1 and determined the effect on PE-induced hypertrophy. Expression of Lef-1 had no significant effect on PE-induced hypertrophy, compatible with ␤-catenin being the limiting factor (Fig. 4D). In contrast, expression of Lef-1⌬␤-cat abrogated the PE-induced increase in cell size (Fig. 4D). To determine whether ␤-catenin might have a role in cardiomyocyte growth in vivo, we performed adenoviral gene transfer in the intact rat heart. At8dafterinjection, cardiomyocytes expressing ␤-catenin⌬ were significantly larger than those transduced with a control virus (AdGFP) or nontransduced cells (Fig. 4E). Mild inflammation was observed in myocardial sections when counterstained with hematoxylin. However, the fact that there was a significant increase in cell size in only the ␤-catenin⌬- transduced cardiomyocytes indicates that paracrine effects from cytokines released by inflammatory cells could not have ac- counted for the changes noted. These data confirm that ␤-cate- Fig. 4. ␤-Catenin is sufficient and necessary for cardiomyocyte hypertrophy. (A) NRVMs were transduced with AdGFP (GFP), Ad␤-catenin (␤-cat), or Ad␤-catenin⌬ nin is both sufficient and necessary for the growth response of (␤-cat⌬), each with a multiplicity of infection of 100 plaque-forming units per cell. NRVMs to hypertrophic stimuli in vitro and sufficient to induce Lysates were blotted with anti-␤-catenin antibody (Upper) or were subjected to cardiomyocyte growth in vivo. immunoprecipitation with anti-vesicular stomatitis virus antibody followed by blotting with anti-␤-catenin antibody (Lower). (B) NRVMs were transduced with Discussion AdGFP, Ad␤-catenin, Ad␤-catenin⌬, or AdNF-AT⌬ at the multiplicities of infec- ␤-Catenin is best known for its role in development and cancer (7). 3 tion noted. Incorporation of [ H]leucine was determined 48 h later. As a refer- To our knowledge, its role in terminally differentiated cells or in ence, [3H]leucine incorporation was determined in AdGFP-transduced cells stimulated with endothelin-1 or PE for 48 h. Values for AdGFP-transduced cells normal tissues of adult mammals has not been explored (11). ϭ Herein, we have identified a biological role for the transactivating treated with vehicle were normalized to 1 (n 5–6 experiments, each done in ␤ triplicate). *, P Ͻ 0.01 vs. AdGFP-transduced cells treated with vehicle. (C Left), activity of -catenin in terminally differentiated cells, and that role ␤-Catenin⌬ induces an increase in cardiomyocyte size in vitro. NRVMs on cover- is as a regulator of hypertrophic stress-induced cardiomyocyte slips were transduced with AdGFP, Ad␤-catenin, or Ad␤-catenin⌬ at a multiplicity growth. Furthermore, the studies identify mechanisms of regulation of infection of 100 plaque-forming units per cell. AdGFP-transduced cells were of ␤-catenin in cultured cardiomyocytes and in vivo in response to stimulated with PE or vehicle (-) for 48 h. Transduced cells were identified by GFP positivity, and cardiomyocyte CSA was determined on images of anti-␣-actinin- stained myocytes (n ϭ 4 experiments; Ն75 myocytes measured per experiment). *, P Ͻ 0.01 vs. AdGFP-transduced cells treated with vehicle. (Right) Inhibition of and D) The same fields stained with FITC-conjugated phalloidin. (Right) Graph ␤-catenin⌬-induced hypertrophy by Lef-1⌬␤-catenin. NRVMs were transduced compares CSA of cells successfully transfected with either Lef-1 or Lef-1⌬␤-cat with AdGFP or Ad␤-catenin⌬ and then were transfected with plasmids encoding with cells that were not transfected (NT), after stimulation with PE or vehicle Myc epitope-tagged wild-type Lef-1 or Lef-1⌬␤-catenin. Cardiomyocyte CSA was (control). Data are from four experiments; Ն75 myocytes measured per ex- determined 48 h later in cells successfully transfected with either Lef-1 or Lef- periment. *, P Ͻ 0.01 vs. CSA of all controls. #, P Ͻ 0.01 vs. CSA of Lef-1⌬␤- 1⌬␤-catenin (Lef-1⌬) and in cells on the same coverslips that had undergone the cat-transfected cells. (Bar, 20 ␮m.) (E) ␤-Catenin⌬ increased cardiomyocyte size transfection protocol but were not successfully transfected (NT; n ϭ 4 experi- in vivo.(Left) AdGFP or Ad␤-catenin⌬ were injected directly into the left ments; Ն75 myocytes measured per experiment). #, P Ͻ 0.01 vs. CSA of Ad␤-cat⌬- ventricular myocardium of rats. Eight days, later cardiomyocyte CSA was transduced cells that were transfected with Lef-1⌬␤-cat. *, P Ͻ 0.01 vs. CSA of all determined on GFP-positive myocytes infected with Ad␤-catenin⌬ (red ar- AdGFP-transduced cells. (D) Inhibition of PE-induced hypertrophy by Lef-1⌬␤- rows) and adjacent nontransduced myocytes (black arrows). (Bar, 20 ␮m.) catenin. (Left) NRVMs were transfected with plasmids encoding Myc-tagged (Right) Graph compares CSA of cells successfully transduced with Ad␤- wild-type Lef-1 (A and B) or Myc-tagged Lef-1⌬␤-cat (C and D). Twenty-four hours catenin⌬, AdGFP, or cells that were not transduced (NT; n ϭ 4 hearts per later, cells were stimulated with PE. Cardiomyocyte CSA was determined 48 h treatment group; Ն50 myocytes measured per heart). *, P Ͻ 0.01 vs. AdGFP later. (A and C) Immunostained with anti-Myc tag to identify transfected cells. (B transduced.

4614 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0835895100 Haq et al. Downloaded by guest on September 30, 2021 stimuli other than Wnts. We found striking differences between the complex, and, although this recruitment was not necessary for mechanisms regulating cytosolic ␤-catenin levels in cardiomyocytes stabilization of ␤-catenin, it did augment Wnt-induced stabili- exposed to hypertrophic stress and those reported previously from zation. Thus, hypertrophic stimuli and Wnt signaling lead to an studies in Drosophila, Xenopus, and in mammalian cells stimulated identical outcome stabilization of ␤-catenin; and, although dif- with Wnts (11). We found that what is believed to be an important ferent mechanisms are used, recruitment of PKB to the Axin mechanism of inhibition of GSK-3 used by Wnt signaling, complex complex may be a common thread of both signaling systems. formation of GSK-3␤ with Frat1, was not a major factor after The cyclin D1 gene is a known target of ␤-catenin (33). hypertrophic stress (21). In fact, we found that hypertrophic stress Although gene transfer of ␤-catenin led to increased cyclin D1 disrupted the GSK-3␤͞Frat1 complex, releasing GSK-3 from this expression in cycling cells, cyclin D1 was not induced in cardi- inhibitor. Hypertrophic stress induced the formation of a multipro- omyocytes (data not shown). The inability of terminally differ- tein complex that included Axin, GSK-3␤, and ␤-catenin. Surpris- entiated cardiomyocytes to up-regulate cyclin D1 might, in part, ingly, the stabilization of the ␤-catenin-degradation complex, which explain the differences in the growth responses observed be- is normally associated with destabilization of ␤-catenin (31), was tween these cells and cells with the ability to proliferate, associated with increased cytosolic levels (or stabilization) of highlighting a possible point of divergence in the regulation of ␤-catenin (Fig. 1 A and C). Our data demonstrating the continued hyperplastic vs. hypertrophic growth potential. presence of ␤-catenin in the Axin complex suggest that release of It has been unclear how hypertrophic stimuli, which primarily ␤-catenin from the complex, which occurs with Wnt signaling produce transient activation (or inhibition) of cellular signaling possibly mediated by Dishevelled (Dvl), casein kinase I␧͞␦ (31), or pathways, can produce the profound and long-term alterations in by GSK-3 inhibition, is not the primary mechanism contributing to gene expression that culminate in the hypertrophic response. ␤͞␤ its stabilization. Rather, our data suggest that the stabilization of The delayed (and prolonged) recruitment of the GSK-3 - ␤-catenin is achieved via Ser-9 phosphorylation of GSK-3␤, and catenin module demonstrated here suggests one mechanism that this, in turn, occurs via recruitment of PKB to the Axin whereby signaling networks may be sequentially recruited to complex. allow the full expression of the hypertrophic phenotype. ␤ We have shown that ␤-catenin is stabilized by hypertrophic Ding et al. (17), studying regulation of -catenin by stimuli ␤ other than Wnts, reported that PKB-induced Ser-9 phosphory- stimuli and that inhibition of GSK-3 , via phosphorylation of Ser-9 ␤ by PKB, appears to be the mechanism by which ␤-catenin is lation (and inhibition) of GSK-3 in response to insulin was ␤ insufficient to stabilize ␤-catenin. In contrast, we have found that stabilized. -Catenin is both sufficient to induce hypertrophic insulin-like growth factor-1 and insulin, which also induce hy- growth when expressed in cardiomyocytes in vitro and in vivo, and pertrophic growth in cardiomyocytes, lead to a biphasic pattern its transcriptional activating activity is necessary for the hypertro- ␤ phic response to physiologically relevant stimuli. These studies of stabilization of -catenin that is similar to that induced by PE ␤ (data not shown). Although we have not clarified the mechanism implicate -catenin as a substrate of GSK-3 that regulates growth of stabilization of ␤-catenin by insulin like growth factor-1, the of terminally differentiated cells and identify a new Wnt- independent mechanism of regulation of the ␤-catenin-degradation findings suggest that the different conclusions reached by Ding complex that is triggered by hypertrophic stimuli acting via mem- et al. (17) may be because of the fact that stabilization of bers of the heterotrimeric G protein-coupled receptor superfamily. ␤-catenin by hypertrophic agonists is restricted to certain cell types, cardiomyocytes being one. ␤ ␤ We thank Paul Hamel, Jeff Molkentin, and Jeffrey Rubin for reagents Wnts also stabilize -catenin via inhibition of GSK-3 , but, in and invaluable advice. This work was supported by the Wellcome Trust contrast to hypertrophic signaling, this is not believed to involve (064925͞Z͞01͞Z to S.H.), a Scientist Development Grant and Estab- PKB-induced Ser-9 phosphorylation (16, 32). Fukumoto et al. lished Investigator Award from the American Heart Association, and the (18) have reported Wnt-induced recruitment of PKB to the Axin National Institutes of Health (Grants HL61688 and HL67371).

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Haq et al. PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4615 Downloaded by guest on September 30, 2021