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Here, we show that entactin-2 expression is strongly, but transiently, induced in myogenic differentiation. Treatment of C2C12 myoblasts with actinomycin D in parallel to the induction of differentiation could demonstrate that this is due to enhanced transcription of the entactin-2 gene. Furthermore, treatment with the translation inhibitor cycloheximide could show that entactin-2 is a primary response gene. As p38 MAP kinase is an important regulator of myogenic differentiation, we also analyzed the possibility that entactin-2 might be a target of this pathway. However, using various p38 MAPK inhibitors, we could not detect involvement of p38 in entactin-2 up-regulation. Most remarkably, expression of the entactin-2 homolog entactin-1 dramatically declined in myogenesis, suggesting different functions of the two entactins in this process. A similar effect was seen in primary myoblasts isolated from two different mouse strains. Expression of high levels of entactin-1 in myoblasts using a retroviral expression system led to a higher proliferation rate both in growth and in differentiation medium and to reduced expression of various myogenic differentiation markers after the induction of differentiation. Furthermore, decreased expression of the entactin-2 gene after treatment of the cells with ent-2-specific siRNA preparation led to reduced expression of the cell cycle inhibitor p21. These data suggest important and distinct functions of entactin-1 and -2 in myogenic differentiation. [ABSTRACT FROM AUTHOR]

Transforming growth factor β1 (TGF-β1) is an important modulator of skin morphogenesis and cutaneous wound repair. To gain insight into the mechanisms of TGF-β1 action in the skin, we used the differential display RT-PCR technique to identify genes that are regulated by this factor in cultured human keratinocytes. We obtained several partial cDNA clones. One of them was identical to the 3'-end of p11, the small and regulatory subunit of the calpactin I complex [(annexin II)2(p11)2]. RNase protection and northern blot analysis revealed specific regulation of expression of both subunits of this heterotetrameric protein (p11 and annexin II) by TGF-β1 as well as by other growth factors, although the time course and degree of induction or suppression were different for each gene. Furthermore, we analyzed p11 and annexin II expression in normal and wounded skin. Both p11 and annexin II mRNAs were found in the dermal and epidermal compartments of normal human skin. Immunohistochemical studies demonstrated the presence of p11 at equally high levels in all layers of normal epidermis and in the hyper-proliferative epithelium at the wound edge. By contrast, annex, in II expression was high in the basal layer of normal epidermis but low in the suprabasal layers and in the hyper-proliferative epithelium at the wound edge, suggesting a differentiation-specific regulation of this calpaetin I subunit. The differential expression and regulation of p11 and annexin II subunits in keratinocytes suggest the existence of different ratios of monomeric versus p11-complexed annexin II that might be associated with different cellular functions. [ABSTRACT FROM AUTHOR]

skNAC (skeletal and heart muscle-specific variant of nascent polypeptide-associated complex) and Smyd1 (SET and MYND domain-containing 1) form a protein dimer which is specific for striated muscle cells. Its function is largely unknown. On the one hand, skNAC-Smyd1 appears to control transcriptional processes in the nucleus, on the other hand, specifically at later stages of myogenic differentiation, both proteins translocate to the sarcoplasm and at least Smyd1 specifically associates with sarcomeric structures and might control myofibrillogenesis and/or sarcomere architecture. Here, using immunofluorescence and electron microscopy, we analyzed sarcomere formation and myofibril organization after siRNA-mediated knockdown of skNAC or Smyd1 expression in murine C2C12 skeletal muscle cells. We found that inhibition of skNAC or Smyd1 expression indeed prevents myofibrillogenesis and sarcomere formation, leading to a disorganized array of myofilaments predominantly within the region immediately beneath the plasma membrane. [ABSTRACT FROM AUTHOR]

The ZFP36 family of zinc finger proteins, including ZFP36, ZFP36L1, and ZFP36L2, regulates the production of growth factors and cytokines via destabilization of the respective mRNAs. We could recently demonstrate that in cultured keratinocytes, expression of the ZFP36 , ZFP36L1 , and ZFP36L2 genes is induced by growth factors and cytokines and that ZFP36L1 is a potent regulator of keratinocyte VEGF production. We now further analyzed the localization and function of ZFP36 proteins in the skin, specifically in epidermal keratinocytes. We found that in human epidermis, the ZFP36 protein could be detected in basal and suprabasal keratinocytes, whereas ZFP36L1 and ZFP36L2 were expressed mainly in the basal layer, indicating different and non-redundant functions of the three proteins in the epidermis. Consistently, upon inhibition of ZFP36 or ZFP36L1 expression using specific siRNAs, there was no major effect on expression of the respective other gene. In addition, we demonstrate that both ZFP36 and ZFP36L1 influence keratinocyte cell cycle, differentiation, and apoptosis in a distinct manner. Finally, we show that similarly as ZFP36L1, ZFP36 is a potent regulator of keratinocyte VEGF production. Thus, it is likely that both proteins regulate angiogenesis via paracrine mechanisms. Taken together, our results suggest that ZFP36 proteins might control reepithelialization and angiogenesis in the skin in a multimodal manner. [ABSTRACT FROM AUTHOR]

Abstract: NAC (nascent polypeptide associated complex) is a heterodimer consisting of an α and a β subunit. skNAC (skeletal and heart muscle-specific form of αNAC) is a variant of αNAC, which is induced in muscle differentiation and regeneration. We show here that skNAC expression is regulated by p38 MAPK, which plays a crucial role in myogenesis. Furthermore, inhibition of skNAC expression in myoblasts via specific siRNAs might lead to disturbed incorporation of myosin heavy chain (MyHC) into sarcomeres, however, it has no inhibitory effect on absolute MyHC protein levels. Taken together, our data suggest that skNAC regulates specific aspects of myogenesis. [Copyright &y& Elsevier]

Abstract: RIP2 is an important regulator of myoblast proliferation and differentiation. We have previously demonstrated that in the myoblast cell line C2C12 and in primary myoblasts, downregulation of rip2 gene expression is a prerequisite for differentiation. To further study the role of rip genes in myogenesis, we compared expression patterns of rip1–4 in two myoblast cell lines, C2C12 and C2F3, after the induction of differentiation. These two cell lines are derived from the same clonal origin, but differ with respect to their differentiation behaviour: specifically, the differentiation process is slower and more incomplete in C2F3 cells. When analyzing cells up to 4 days after the induction of differentiation, we found no downregulation of rip2 gene expression in C2F3 cells, which might be linked to the low differentiation potential of these cells. In addition, in contrast to C2C12 cells, the rip3 gene was not expressed in C2F3 cells. To further study the role of rip genes in the regulation of myoblast growth and differentiation, we analyzed expression patterns of rip1–4 in rhabdomyosarcoma cell lines. We found that in these cells, rip2 expression was not downregulated after the induction of differentiation. Furthermore, in contrast to normal myoblasts, they did not express the rip3 and rip4 genes. Thus, we focused on the functional role of RIP2 in rhabdomyosarcoma cells. Inhibition of rip2 gene expression in C2C12 and in rhabdomyosarcoma cells using specific siRNAs led to decreased proliferation and promoted the differentiation process of these cells. These data indicate that differential expression of rip genes can be associated with abnormal growth and differentiation behaviour of skeletal myoblasts. [Copyright &y& Elsevier]

Abstract: TIS11B is a zinc-finger protein of the tristetraprolin (TTP) family. Using cDNA microarray analysis, we could identify the Tis11B gene based on its differential expression in myogenesis. Here, we demonstrate that expression of the Tis11B gene is strongly induced during differentiation of the murine myoblast cell line C2C12. By contrast, expression of Ttp itself was not induced in myogenesis. Pretreatment of the cells with the translation inhibitor cycloheximide demonstrated that Tis11B was a primary response gene in this process. In addition, pretreatment with the transcription inhibitor actinomycin D demonstrated that gene expression was regulated at the transcriptional level. Since specific inhibitors of p38 MAP kinase completely blocked Tis11B induction, we conclude that expression of the Tis11B gene is regulated at least in part by this signaling pathway which plays a central role in myogenesis. Induction of Tis11B expression was also observed in primary myoblasts isolated from two different mouse strains, indicating physiological relevance of our results. In addition, TIS11B might also be an important player during myogenic differentiation and regeneration in vivo, as we detected a marked decrease in expression in several muscle tissues of the dystrophic mdx mouse, a model for continuous muscle degeneration and regeneration. These data suggest that TIS11B is an important regulator of myogenesis. [Copyright &y& Elsevier]

Receptor-interacting proteins (RIPs) are important regulators of cell proliferation and differentiation. As RIP4 is a crucial modulator of epidermal differentiation, we analyzed the expression of different rip genes in healing skin wounds. Rip4 expression was strongly downregulated in keratinocytes of the hyperproliferative epithelium at the wound edge early after injury and only returned to basal levels after completion of wound repair. Rip3 expression was strongly induced as early as 1 day after wounding. In contrast, rip and rip2 expression remained unaltered. To determine the factors that regulate rip4 gene expression in keratinocytes, human HaCaT keratinocytes were used as a model system. We found that scratch wounding as well as treatment with whole serum, phorbol esters, the growth/differentiation factors epidermal growth factor, transforming growth factor-β, and activin A, or the proinflammatory cytokines tumor necrosis factor-α and IL-1β strongly suppressed rip4 expression in these cells. In contrast, the steroid dexamethasone and all-trans retinoic acid slightly stimulated rip4 expression. Suppression of rip4 expression in keratinocytes using small interfering RNA technology reduced the activation of NF-κB, and enhanced the expression of epidermal differentiation markers in these cells. These data suggest important and unique functions of different RIP proteins in keratinocytes of normal and wounded skin.Journal of Investigative Dermatology (2007) 127, 538–544. doi:10.1038/sj.jid.5700588; published online 12 October 2006 [ABSTRACT FROM AUTHOR]