Apoptosis repressor with caspase recruitment domain (ARC) inhibits myogenic differentiation

Apoptosis repressor with caspase recruitment domain (ARC), an anti-apoptotic protein, is highly expressed in differentiated heart and skeletal muscle. Apoptosis and differentiation share numerous common pathways; therefore, we examined the impact of ARC on H9c2-myoblast differentiation. We demonstrate that ARC expression levels increase and stabilize upon differentiation. ARC-overexpression in pre-differentiated H9c2-cells suppresses differentiation; indicated by increased myotube formation, nuclear fusion and expression of the differentiation markers myogenin and troponin-T. ARC-overexpression inhibited myoblast differentiation associated caspase-3 activation, suggesting ARC inhibits myogenic differentiation through caspase inhibition. In summary, we show a novel role for ARC in the regulation of muscle differentiation.     

S100B inhibits myogenic differentiation and myotube formation in a RAGE-independent manner

S100B is a Ca(2+)-modulated protein of the EF-hand type with both intracellular and extracellular roles. S100B, which is most abundant in the brain, has been shown to exert trophic and toxic effects on neurons depending on the concentration attained in the extracellular space. S100B is also found in normal serum, and its serum concentration increases in several nervous and nonnervous pathological conditions, suggesting that S100B-expressing cells outside the brain might release the protein and S100B might exert effects on nonnervous cells. We show here that at picomolar to nanomolar levels, S100B inhibits myogenic differentiation of rat L6 myoblasts via inactivation of p38 kinase with resulting decrease in the expression of the myogenic differentiation markers, myogenin, muscle creatine kinase, and myosin heavy chain, and reduction of myotube formation. Although myoblasts express the multiligand receptor RAGE, which has been shown to transduce S100B effects on neurons, S100B produces identical effects on myoblasts overexpressing either full-length RAGE or RAGE lacking the transducing domain. This suggests that S100B affects myoblasts by interacting with another receptor and that RAGE is not the only receptor for S100B. Our data suggest that S100B might participate in the regulation of muscle development and regeneration by inhibiting crucial steps of the myogenic program in a RAGE-independent manner.     

The homeodomain protein Barx2 promotes myogenic differentiation and is regulated by myogenic regulatory factors

The homeobox protein Barx2 is expressed in both smooth and skeletal muscle and is up-regulated during differentiation of skeletal myotubes. Here we use antisense-oligonucleotide inhibition of Barx2 expression in limb bud cell culture to show that Barx2 is required for myotube formation. Moreover, overexpression of Barx2 accelerates the fusion of MyoD-positive limb bud cells and C2C12 myoblasts. However, overexpression of Barx2 does not induce ectopic MyoD expression in either limb bud cultures or in multipotent C3H10T1/2 mesenchymal cells, and does not induce fusion of C3H10T1/2 cells. These results suggest that Barx2 acts downstream of MyoD. To test this hypothesis, we isolated the Barx2 gene promoter and identified DNA regulatory elements that might control Barx2 expression during myogenesis. The proximal promoter of the Barx2 gene contained binding sites for several factors involved in myoblast differentiation including MyoD, myogenin, serum response factor, and myocyte enhancer factor 2. Co-transfection experiments showed that binding sites for both MyoD and serum response factor are necessary for activation of the promoter by MyoD and myogenin. Taken together, these studies indicate that Barx2 is a key regulator of myogenic differentiation that acts downstream of muscle regulatory factors.     

Intracellular localisation and expression of mammalian CDC2 protein during myogenic differentiation

Myogenic differentiation involves withdrawal of myoblasts from the cell cycle and fusion to form multinucleate myotubes. To examine the role that cell cycle control genes may play in this process, we investigated the steady state levels of CDC2 protein and RNA during myogenesis of L6E9 rat myoblasts. Indirect immunofluorescence using a CDC2 affinity-purified antibody showed that this protein is localised exclusively in the cytoplasm with a higher concentration perinuclearly. Both protein and RNA levels were down-regulated to similar extents early in the differentiation process, as cells became quiescent. There was a further down-regulation of protein after fusion to form myotubes. Autonomous expression of CDC2 protein in L6E9 cells, after stable transfection with a metallothionein: CDC2 gene construct, failed to inhibit the differentiation process. This suggests that, although there is down-regulation in levels of CDC2 RNA and protein during myogenesis, this phenomenon per se does not play a primary role in controlling the differentiation process. If CDC2 is involved in control of differentiation, this must depend on post-translational modification of the protein.     

Cytohesin-associated scaffolding protein (CASP) is a substrate for granzyme B and ubiquitination

Natural killer (NK) cells are a sub-population of cytotoxic lymphocytes that can kill tumor or infected cells without prior exposure, by secreting the contents of preformed cytotoxic vesicles, containing perforin and granzymes, at the immune synapse. Cytohesin-associated scaffolding protein (CASP) is an adaptor molecule uniquely expressed in lymphocytes that forms complexes with both vesicle-initiating and sorting proteins, and has roles in NK cell migration, cytotoxicity, and cytokine secretion. In this study, we show that CASP contains a conserved granzyme B cleavage site that could modify its intracellular localization and interaction with sorting nexin 27. We also provide evidence that CASP is modified by ubiquitination. Both of these post-translational modifications could rapidly modify CASP function and highlight the dynamic regulatory mechanisms that direct its role in NK cell functions.     

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.     

The X protein of hepatitis B virus binds to the F box protein Skp2 and inhibits the ubiquitination and proteasomal degradation of c-Myc

The HBx protein of hepatitis B virus is involved in deregulation of cell cycle and development of hepatocellular carcinoma. Since c-Myc also plays an important role in cell proliferation and tumor development, we studied its regulation by HBx in a human hepatoma cell line. Co-expression of HBx and c-Myc resulted in increased stability of intracellular c-Myc. HBx blocked the ubiquitination of Myc through a direct interaction with the F box region of Skp2 and destabilization of the SCF(Skp2) complex. We suggest that sustained presence of c-Myc combined with mitogenic activity inherent to HBx may be associated with cell cycle deregulation and transformation.     

Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro

The in vitro effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on osteogenic and myogenic differentiation was examined in two clonal cell lines of rat osteoblast-like cells at different differentiation stages, ROB-C26 (C26) and ROB-C20 (C20). The C26 is a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes; the C20 is a more differentiated osteoblastic cell line. Proliferation was stimulated by rhBMP-2 in C26 cells, but inhibited in C20 cells. rhBMP-2 greatly increased alkaline phosphate (ALP) activity in C26 cells, but not in C20 cells. The steady-state level of ALP mRNA was also increased by rhBMP-2 in C26 cells, but not in C20 cells. Production of 3',5'-cAMP in response to parathyroid hormone (PTH) was dose-dependently enhanced by adding rhBMP-2 in both C26 and C20 cells, though the stimulatory effect was much greater in the former. There was neither basal expression of osteocalcin mRNA nor its protein synthesis in C26 cells, but they were strikingly induced by rhBMP-2 in the presence of 1 alpha,25-dihydroxyvitamin D3. rhBMP-2 induced no appreciable changes in procollagen mRNA levels of type I and type III in the two cell lines. Differentiation of C26 cells into myotubes was greatly inhibited by adding rhBMP-2. The inhibitory effect of rhBMP-2 on myogenic differentiation was also observed in clonal rat skeletal myoblasts (L6). Like BMP-2, TGF-beta 1 inhibited myogenic differentiation. However, unlike BMP-2, TGF-beta 1 decreased ALP activity in both C26 and C20 cells. TGF-beta 1 induced neither PTH responsiveness nor osteocalcin production in C26 cells, but it increased PTH responsiveness in C20 cells. These results clearly indicate that rhBMP-2 is involved, at least in vitro, not only in inducing differentiation of osteoblast precursor cells into more mature osteoblast-like cells, but also in inhibiting myogenic differentiation.     

A novel muscle-specific beta 1 integrin binding protein (MIBP) that modulates myogenic differentiation

Myogenesis is regulated by cell adhesion receptors, including integrins of the beta1 family. We report the identification of a novel muscle-specific beta1 integrin binding protein (MIBP). MIBP binds to the membrane-proximal cytoplasmic region shared by beta1A and beta1D integrins, and the binding occurs in vivo as well as in vitro. Furthermore, we show that MIBP is abundantly expressed by C2C12 myogenic cells before fusion, and the expression of MIBP is dramatically downregulated during subsequent differentiation. Finally, we show that overexpression of MIBP in C2C12 cells resulted in a suppression of fusion and terminal differentiation, suggesting that MIBP may play a key role in controlling the progression of muscle differentiation.     

Mechanism of activation of protein kinase D2(PKD2) by the CCK(B)/gastrin receptor

Recently, we cloned a novel serine/threonine kinase termed protein kinase D2 (PKD2). PKD2 can be activated by phorbol esters both in vivo and in vitro but also by gastrin via the cholecystokinin/CCK(B) receptor in human gastric cancer cells stably transfected with the CCK(B)/gastrin receptor (AGS-B cells). Here we identify the mechanisms of gastrin-induced PKD2 activation in AGS-B cells. PKD2 phosphorylation in response to gastrin was rapid, reaching a maximum after 10 min of incubation. Our data demonstrate that gastrin-stimulated PKD2 activation involves a heterotrimeric G alpha(q) protein as well as the activation of phospholipase C. Furthermore, we show that PKD2 can be activated by classical and novel members of the protein kinase C (PKC) family such as PKC alpha, PKC epsilon, and PKC eta. These PKCs are activated by gastrin in AGS-B cells. Thus, PKD2 is likely to be a novel downstream target of specific PKCs upon the stimulation of AGS-B cells with gastrin. Our data suggest a two-step mechanism of activation of PKD2 via endogenously produced diacylglycerol and the activation of PKCs.     

A novel deletion mutation of mouse ruby-eye 2 named ru2(d)/Hps5(ru2-d) inhibits melanocyte differentiation and its impaired differentiation is rescued by L-tyrosine

In our laboratory, a single autosomal recessive mutation in a phenotype similar to ruby-eye (ru/Hps6(ru)) or ruby-eye 2 (ru2/Hps5(ru2)) spontaneously occurred in siblings of C57BL/10JHir (+/+, black) mice in 2006. RT-PCR analysis revealed that this novel mutation, named ru2(d)/Hps5(ru2-d), exhibited frameshift by 997G deletion in the Hps5 gene. To clarify the mechanism of the hypopigmentation, the characteristics of proliferation and differentiation of ru2(d)/ru2(d) epidermal melanoblasts and melanocytes cultured in a serum-free medium were investigated. The proliferation of ru2(d)/ru2(d) melanoblasts and melanocytes did not differ from that of +/+ melanoblasts and melanocytes. However, the differentiation of ru2(d)/ru2(d) melanocytes was greatly inhibited. Tyrosinase (TYR) activity, expression of TYR, TYR-related protein 1 (TRP1) and TRP2 (dopachrome tautomerase, DCT), eumelanin synthesis, and the number of stage IV melanosomes markedly decreased in ru2(d)/ru2(d) melanocytes. However, excess L-tyrosine (Tyr) added to culture media from initiation of the primary culture rescued the reduced differentiation through increase in TYR activity, expression of TYR, TRP1, TRP2 and Kit, eumelanin synthesis, and stage IV melanosomes. L-Tyr injected into ru2(d)/ru2(d) mice also stimulated melanocyte differentiation. These results suggest that the ru2(d) allele inhibits melanocyte differentiation, and that its impaired differentiation is rescued by excess Tyr.     

Ni(II) affects ubiquitination of core histones H2B and H2A

The molecular mechanisms of nickel-induced malignant cell transformation include effects altering the structure and covalent modifications of core histones. Previously, we found that exposure of cells to Ni(II) resulted in truncation of histones H2A and H2B and thus elimination of some modification sites. Here, we investigated the effect of Ni(II) on one such modification, ubiquitination, of histones H2B and H2A in nuclei of cultured 1HAEo- and HPL1D human lung cells. After 1-5 days of exposure, Ni(II) up to 0.25 mM stimulated mono-ubiquitination of both histones, while at higher concentrations a suppression was found. Di-ubiquitination of H2A was not affected except for a drop after 5 days at 0.5 mM Ni(II). The decrease in mono-ubiquitination coincided with the appearance of truncated H2B that lacks the K120 ubiquitination site. However, prevention of truncation did not avert the decrease of H2B ubiquitination, indicating mechanistic independence of these effects. The changes in H2B ubiquitination did not fully coincide with concurrent changes in the nuclear levels of the ubiquitin-conjugating enzymes Rad6 and UbcH6. Overall, our results suggest that dysregulation of H2B ubiquitination is a part of Ni(II) adverse effects on gene expression and DNA repair which may assist in cell transformation.     

K restriction inhibits protein phosphatase 2B (PP2B) and suppression of PP2B decreases ROMK channel activity in the CCD

We used Western blot analysis to examine the effect of dietary K intake on the expression of serine/threonine protein phosphatase in the kidney. K restriction significantly decreased the expression of catalytic subunit of protein phosphatase (PP)2B but increased the expression of PP2B regulatory subunit in both rat and mouse kidney. However, K depletion did not affect the expression of PP1 and PP2A. Treatment of M-1 cells, mouse cortical collecting duct (CCD) cells, or 293T cells with glucose oxidase (GO), which generates superoxide anions through glucose metabolism, mimicked the effect of K restriction on PP2B expression and significantly decreased expression of PP2B catalytic subunits. However, GO treatment increased expression of regulatory subunit of PP2B and had no effect on expression of PP1, PP2A, and protein tyrosine phosphatase 1D. Moreover, deletion of gp91-containing NADPH oxidase abolished the effect of K depletion on PP2B. Thus superoxide anions or related products may mediate the inhibitory effect of K restriction on the expression of PP2B catalytic subunit. We also used patch-clamp technique to study the effect of inhibiting PP2B on renal outer medullary K (ROMK) channels in the CCD. Application of cyclosporin A or FK506, inhibitors of PP2B, significantly decreased ROMK channels, and the effect of PP2B inhibitors was abolished by blocking p38 mitogen-activated protein kinase (MAPK) and ERK. Furthermore, Western blot demonstrated that inhibition of PP2B with cyclosporin A or small interfering RNA increased the phosphorylation of ERK and p38 MAPK. We conclude that K restriction suppresses the expression of PP2B catalytic subunits and that inhibition of PP2B decreases ROMK channel activity through stimulation of MAPK in the CCD.     

Mechano growth factor (MGF) promotes proliferation and inhibits differentiation of porcine satellite cells (PSCs) by down-regulation of key myogenic transcriptional factors

Porcine satellite cells represent an ideal model system for studying the cellular and molecular basis regulating myogenic stem cell proliferation and differentiation and for exploring the experimental conditions for myoblast transplantation. Here, we investigated the effects of mechano growth factor (MGF), a spliced variant of the IGF-1 gene, on porcine satellite cells. We show that MGF potently stimulated proliferation while inhibited differentiation of porcine satellite cells. MGF-treatment acutely down-regulates the expression of myogenic determination factor (MyoD) and the cyclin-dependent kinase inhibitor p21. MGF-treatment also markedly reduced the overall expression of cyclin B1 and key factors of the myogenic regulatory and myocyte enhancer families, including Myogenein and MEF2A. Taken together, the gene expression data from MGF-treated porcine satellite cells are in favor of a molecular model in which MGF inhibits porcine satellite cell differentiation by down-regulating either the activity or expression of MyoD, which, in turn, suppresses the expression of key genes required for cell cycle progression and differentiation, such as p21, Myogenin, and MEF2. Overall, our findings are in support of the previous suggestion that MGF may be used in vivo and in vitro to promote proliferation of myogenic stem cells to prevent and treat age-related muscle degenerative diseases.