Dataset Information

Yes-associated protein activation potentiates glycogen synthase kinase-3 inhibitor-induced proliferation of neonatal cardiomyocytes and iPS cell-derived cardiomyocytes.

ABSTRACT: Because mammalian cardiomyocytes largely cease to proliferate immediately after birth, the regenerative activity of the heart is limited. To date, much effort has been made to clarify the regulatory mechanism of cardiomyocyte proliferation because the amplification of cardiomyocytes could be a promising strategy for heart regenerative therapy. Recently, it was reported that the inhibition of glycogen synthase kinase (GSK)-3 promotes the proliferation of neonatal rat cardiomyocytes (NRCMs) and human iPS cell-derived cardiomyocytes (hiPSC-CMs). Additionally, Yes-associated protein (YAP) induces cardiomyocyte proliferation. The purpose of this study was to address the importance of YAP activity in cardiomyocyte proliferation induced by GSK-3 inhibitors (GSK-3Is) to develop a novel strategy for cardiomyocyte amplification. Immunofluorescent microscopic analysis using an anti-Ki-67 antibody demonstrated that the treatment of NRCMs with GSK-3Is, such as BIO and CHIR99021, increased the ratio of proliferative cardiomyocytes. YAP was localized in the nuclei of more than 95% of cardiomyocytes, either in the presence or absence of GSK-3Is, indicating that YAP was endogenously activated. GSK-3Is increased the expression of β-catenin and promoted its translocation into the nucleus without influencing YAP activity. The knockdown of YAP using siRNA or pharmacological inhibition of YAP using verteporfin or CIL56 dramatically reduced GSK-3I-induced cardiomyocyte proliferation without suppressing β-catenin activation. Interestingly, the inhibition of GSK-3 also induced the proliferation of hiPSC-CMs under sparse culture conditions, where YAP was constitutively activated. In contrast, under dense culture conditions, in which YAP activity was suppressed, the proliferative effects of GSK-3Is on hiPSC-CMs were not detected. Importantly, the activation of YAP by the knockdown of α-catenin restored the proproliferative activity of GSK-3Is. Collectively, YAP activation potentiates the GSK-3I-induced proliferation of cardiomyocytes. The blockade of GSK-3 in combination with YAP activation resulted in remarkable amplification of cardiomyocytes.

SUBMITTER: Kametani Y

PROVIDER: S-EPMC9311433 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:RationalePulmonary arterial hypertension (PAH) is a rare progressive pulmonary vascular disorder associated with vascular remodeling and right heart failure. Vascular remodeling involves numerous signaling cascades governing pulmonary arterial smooth muscle cell (PASMC) proliferation, migration and differentiation. Glycogen synthase kinase 3beta (GSK3ß) is a serine/threonine kinase and can act as a downstream regulatory switch for numerous signaling pathways. Hence, we hypothesized that GSK3ß plays a crucial role in pulmonary vascular remodeling.MethodsAll experiments were done with lung tissue or isolated PASMCs in a well-established monocrotaline (MCT)-induced PAH rat model. The mRNA expression of Wnt ligands (Wnt1, Wnt3a, Wnt5a), upstream Wnt signaling regulator genes (Frizzled Receptors 1, 2 and secreted Frizzled related protein sFRP-1) and canonical Wnt intracellular effectors (GSK3ß, Axin1) were assessed by real-time polymerase chain reaction and protein levels of GSK3ß, phospho-GSK3ß (ser 9) by western blotting and localization by immunohistochemistry. The role of GSK3ß in PASMCs proliferation was assessed by overexpression of wild-type GSK3ß (WT) and constitutively active GSK3ß S9A by [(3)H]-thymidine incorporation assay.ResultsIncreased levels of total and phosphorylated GSK3ß (inhibitory phosphorylation) were observed in lungs and PASMCs isolated from MCT-induced PAH rats compared to controls. Further, stimulation of MCT-PASMCs with growth factors induced GSK3ß inactivation. Most importantly, treatment with the PDGFR inhibitor, Imatinib, attenuated PDGF-BB and FCS induced GSK3ß phosphorylation. Increased expression of GSK3ß observed in lungs and PASMC isolated from MCT-induced PAH rats was confirmed to be clinically relevant as the same observation was identified in human iPAH lung explants. Overexpression of GSK3ß significantly increased MCT-PASMCs proliferation by regulating ERK phosphorylation. Constitutive activation of GSK3ß (GSK3ß S9A, 9th serine replaced to alanine) inhibited MCT-PASMCs proliferation by decreasing ERK phosphorylation.ConclusionThis study supports a central role for GSK3ß in vascular remodeling processes and suggests a novel therapeutic opportunity for the treatment of PAH.

Project description:UnlabelledThe Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death.ImportanceKSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.

Dataset Information

Yes-associated protein activation potentiates glycogen synthase kinase-3 inhibitor-induced proliferation of neonatal cardiomyocytes and iPS cell-derived cardiomyocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets