Endogenous expression and localization of myostatin and its relation to MHC distribution in C2C12 skeletal muscle cells

Myostatin is a negative regulator of skeletal muscle growth. We have previously reported that recombinant myostatin protein inhibits DNA and protein synthesis in C2C12 cells. Our objective was to assess if C2C12 cells express myostatin, determine its sub-cellular localization and the developmental stage of C2C12 cells in which myostatin mRNA and protein are expressed. To study the endogenous expression of myostatin, C2C12 myoblasts were allowed to progress to myotubes, and changes in the levels of endogenous myostatin mRNA expression were determined by RT-PCR. The myostatin protein and the two major myosin heavy chain (MHC) isoforms (MHC-I and -II) were determined by Western blot. Confirmation of the relative MHC expression patterns was obtained by a modified polyacrylamide gel electropheretic (PAGE) procedure. Imunofluorescence staining was employed to localize the site of myostatin expression and the relative distribution of the MHC isoforms. Co-expression of these proteins was studied using a dual staining approach. Expression of myostatin mRNA was found in myotubes but not in myoblasts. Myostatin protein was seen in most but not all, of the nuclei of polynucleated fibers expressing MHC-II, and myostatin was detected in the cytoplasm of myotube. The localization of myostatin protein in myotube nuclei was confirmed by Western blot of isolated nuclear and cytoplasmic fractions. Incubation of C2C12 myotubes with graded doses of dexamethasone dose-dependently increased the intensity of nuclear myostatin immunostaining and also resulted in the appearance of cytoplasmic expression. In conclusion, myostatin was expressed mostly in C2C12 myotubes nuclei expressing MHC-II. Its predominant

Cox-2 Promotes Chromogranin A Expression and Bioactivity: Evidence for a PGE2-Dependent Mechanism and the Involvement of a Proximal cAMP-Responsive Element.

Abstract The prostanoid biosynthetic enzyme cyclooxygenase-2 (Cox-2) is upregulated in several neuroendocrine tumors. The aim of the current study was to employ a neuroendocrine cell (PC12) model of Cox-2 over-expression to identify gene products that might be implicated in the oncogenic and/or inflammatory actions of this enzyme in the setting of neuroendocrine neoplasia. Expression array and real-time PCR analysis demonstrated that levels of the neuroendocrine marker chromogranin A (CGA) were 2-fold and 3.2-fold higher, respectively, in Cox-2 over-expressing cells (PCXII) vs their control (PCMT) counterparts. Immunocytochemical and immunoblotting analyses confirmed that both intracellular and secreted levels of CGA were elevated in response to Cox-2 induction. Moreover, exogenous addition of prostaglandin E2 (1u�­M), mimicked this effect in PCMT cells, while treatment of PCXII cells with the Cox-2 selective inhibitor NS-398 (100 nM) reduced CGA expression levels, thereby confirming the biospecificity of this finding. Levels of neurone specific enolase (NSE) were similar in the two cell lines, suggesting that the effect of Cox-2 on CGA expression was specific and not due to a global enhancement of neuroendocrine marker expression/differentiation. Cox-2-dependent CGA upregulation was associated with significantly increased chromaffin granule number and intracellular and secreted levels of dopamine. CGA promoter-driven reporter gene expression studies provided evidence that prostaglandin E2-dependent upregulation required a proximal cAMP-responsive element (CRE; -71 - -64 bp). This study is the first to demonstrate that Cox-2 upregulates both CGA expression and bioactivity in a neuroendocrine cell line and has major implications for the role of this polypeptide in the pathogenesis of neuroendocrine cancers in which Cox-2 is upregulated.