Mirk/Dyrk1B Mediates Survival during the Differentiation of C2C12Myoblasts

Mercer, Stephen E.; Ewton, Daina Z.; Deng, Xiaobing; Lim, Seunghwan; Mazur, Thomas R.; Friedman, Eileen

doi:10.1074/jbc.m413594200

Cited by 88 publications

(119 citation statements)

References 54 publications

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“…Our finding that CIBZ down-regulation by siRNA is sufficient to induce apoptosis in C2C12 and p53 Ϫ/Ϫ MEFs suggests strongly that CIBZ functions as an anti-apoptotic regulator and that overexpressed CIBZ may prevent apoptosis induced by a variety of triggers, in a similar fashion to Mirk (27). However, we found no evidence that ectopic CIBZ expression prevents serum-, UV light-, or staurosporine-induced apoptosis in several cell lines (data not shown).…”

Section: Discussionmentioning

confidence: 65%

Down-regulation of CIBZ, a Novel Substrate of Caspase-3, Induces Apoptosis

Oikawa

Matsuda

Nishii

et al. 2008

Journal of Biological Chemistry

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We previously identified and characterized a murine BTB domain-containing protein, CIBZ (ZBTB38 in human), that interacts with CtBP and binds to methylated CpGs. However, its physiological function remained unknown. As CtBP is reportedly involved in p53-independent programmed cell death, we examine here whether CIBZ is associated with apoptosis. We found that CIBZ was highly expressed in proliferating C2C12 cells but that its expression levels decreased upon induction of apoptosis by serum starvation. Knockdown of CIBZ by small interfering RNA in C2C12 cells induced apoptosis, as determined by an increase of annexin V/propidium iodide labeling, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. CIBZ inhibition also activated caspase-7 and caspase-9, suggesting that CIBZ-associated apoptosis occurs through the mitochondrial pathway. Notably, knockdown of CIBZ in p53 ؊/؊ mouse embryonic fibroblast cells also activated caspase-3 and cleavage of poly(ADP-ribose) polymerase, indicating that CIBZassociated apoptosis is mediated by a p53-independent pathway; however, because both common and distinct targets are regulated by CIBZ-and CtBP-associated apoptosis, we conclude that more than one pathway is involved. Finally, using mutagenesis and an in vitro caspase cleavage assay, we show that CIBZ is a novel substrate of caspase-3 and identify two caspase-3 recognition sites. These findings indicate, collectively, that CIBZ plays an important role by participating in the negative regulation of apoptosis in murine cells.Apoptosis is a genetically controlled form of cell death that plays critical roles during development and tissue homeostasis by ensuring the removal of damaged or unnecessary cells (1). Activation of proteolytic enzymes called caspases is a key step in the apoptotic program. Once the initiator caspase, the best characterized of which is caspase-9, is activated by cellular stress signals, it processes and activates downstream effector caspases such as caspase-3 and caspase-7. For caspase-3, the 32-kDa inactive proenzyme is cleaved to 17-and 12-kDa fragments to form an active heterotetramer. This active form can specifically cleave its substrates at a DXXD motif to induce DNA fragmentation and morphological changes typical of cells undergoing apoptosis (1, 2). One of the major substrates of caspase-3 is poly(ADP-ribose) polymerase (PARP), 3 and cleaved PARP and cleaved caspase-3 itself are regarded as signature markers of apoptosis (2, 3).Murine C2C12 cells are a well established in vitro model system to study apoptosis as well as myogenesis in developing muscle because a significant fraction of C2C12 cells undergo apoptosis, cell cycle withdrawal, and differentiation when cultured with 2% horse serum-containing medium (referred to as differentiation medium (DM)) (4 -7). Growing evidence indicates that the expression of many regulatory proteins is stimulated or suppressed during apoptosis induced by DM. The upregulated proteins include the cyclin-dependent kinase inhibitors p21 and p27 (4, 8...

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Section: Discussionmentioning

confidence: 65%

Down-regulation of CIBZ, a Novel Substrate of Caspase-3, Induces Apoptosis

Oikawa

Matsuda

Nishii

et al. 2008

Journal of Biological Chemistry

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“…Recruitment of cells in G0/G1 stage is a pre-and pro-differentiation arrest of skeletal muscle myoblasts, necessary to the subsequent development of myotubes (Mercer et al 2005). For cell cycle progression, the cellular exit from G1-phase and entry into S-phase require formation and activation of cyclin-cyclin-dependent kinases (CDKs) complexes.…”

Section: Introductionmentioning

confidence: 99%

Actions of 1,25(OH)2-vitamin D3 on the cellular cycle depend on VDR and p38 MAPK in skeletal muscle cells

Irazoqui¹,

Boland²,

Buitrago³

2014

Journal of Molecular Endocrinology

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Previously, we have reported that 1,25(OH) 2 -vitamin D 3 (1,25D) activates p38 MAPK (p38) in a vitamin D receptor (VDR)-dependent manner in proliferative C2C12 myoblast cells. It was also demonstrated that 1,25D promotes muscle cell proliferation and differentiation. However, we did not study these hormone actions in depth. In this study we have investigated whether the VDR and p38 participate in the signaling mechanism triggered by 1,25D. In C2C12 cells, the VDR was knocked down by a shRNA, and p38 was specifically inhibited using SB-203580. Results from cell cycle studies indicated that hormone stimulation prompts a peak of S-phase followed by an arrest in the G0/G1-phase, events which were dependent on VDR and p38. Moreover, 1,25D increases the expression of cyclin D3 and the cyclin-dependent kinase inhibitors, p21Waf1/Cip1 and p27 Kip1 , while cyclin D1 protein levels did not change during G0/G1 arrest. In all these events, p38 and VDR were required. At the same time, a 1,25D-dependent acute increase in myogenin expression was observed, indicating that the G0/G1 arrest of cells is a pro-differentiative event. Immunocytochemical assays revealed co-localization of VDR and cyclin D3, promoted by 1,25D in a p38-dependent manner. When cyclin D3 expression was silenced, VDR and myogenin levels were downregulated, indicating that cyclin D3 was required for 1,25D-induced VDR expression and the concomitant entrance into the differentiation process. In conclusion, the VDR and p38 are involved in control of the cellular cycle by 1,25D in skeletal muscle cells, providing key information on the mechanisms underlying hormone regulation of myogenesis.

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“…Depletion of Mirk by RNAi blocked myoblast survival and increased the activation of caspase-3 (10). Moreover, overexpression of wild-type Mirk depressed apoptosis during muscle differentiation, whereas overexpression of kinase-inactive mutant Mirk had no antiapoptotic activity (10). Mirk/Dyrk1B was also shown to have survival functions in HeLa cervical carcinoma cells in high-throughput screening of the human kinome by RNAi (11).…”

Section: Introductionmentioning

confidence: 99%

Mirk/Dyrk1b Mediates Cell Survival in Rhabdomyosarcomas

et al. 2006

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Rhabdomyosarcoma is the most common sarcoma in children and is difficult to treat if the primary tumor is nonresectable or if the disease presents with metastases. The function of the serine/threonine kinase Mirk was investigated in this cancer. Mirk has both growth arrest and survival functions in terminally differentiating skeletal myoblasts. Maintenance of Mirk growth arrest properties would cause down-regulation of Mirk in transformed myoblasts. Alternatively, Mirk expression would be retained if rhabdomyosarcoma cells used Mirk survival capability. Mirk expression was significant in 12 of 16 clinical cases of rhabdomyosarcoma. Mirk was detected in each rhabdomyosarcoma cell line examined. Mirk was a functional kinase in each of three rhabdomyosarcoma cell lines, where it proved to be more active than in C2C12 skeletal myoblasts. Mirk mediated survival of the majority of clonogenic rhabdomyosarcoma cells. Knockdown of Mirk by RNA interference reduced the fraction of RD and of Rh30 rhabdomyosarcoma cells capable of colony formation 3-to 4-fold in multiple experiments. Depletion of Mirk induced cell death by apoptosis, as shown by increased numbers of terminal deoxynucleotidyl transferase-mediated nick-end labeling-positive cells and by increased binding of Annexin V. Mirk is a stress-activated kinase that mediates expression of contractile proteins in differentiating myoblasts, but Mirk is not essential for muscle formation in the embryo. It is likely that Mirk also facilitates survival of satellite cell-derived rhabdomyoblasts in regenerating skeletal muscle and aids their differentiation. This survival function is maintained in rhabdomyosarcoma, where Mirk may be a novel therapeutic target. (Cancer Res 2006; 66(10): 5143-50)

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Mirk/Dyrk1B Mediates Survival during the Differentiation of C2C12Myoblasts

Cited by 88 publications

References 54 publications

Down-regulation of CIBZ, a Novel Substrate of Caspase-3, Induces Apoptosis

Down-regulation of CIBZ, a Novel Substrate of Caspase-3, Induces Apoptosis

Actions of 1,25(OH)2-vitamin D3 on the cellular cycle depend on VDR and p38 MAPK in skeletal muscle cells

Mirk/Dyrk1b Mediates Cell Survival in Rhabdomyosarcomas

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