Effects of a GSK-3ß inhibitor on the renal expression levels of RANK, RANKL and NF-κB in a rat model of diabetic nephropathy.

The present study aimed to investigate the effects of glycogen synthase kinase-3ß (GSK-3ß) on the expression levels of receptor activator of nuclear factor (NF)-κB (RANK), RANK ligand (RANKL) and NF-κB in the renal tissues of rats modeling diabetic nephropathy (DN). The rats were allocated at random into three groups, as follows: Normal control group (NC), the DN model group (DNM group) and the DN model lithium chloride (LiCl) intervention group (DNI group). Urinary proteins were examined by staining with the Coomassie Brilliant Blue dye for 24 h. Histochemical analyses of kidney tissue sections were conducted using hematoxylin and eosin staining, after which the kidney pathology of the rats was observed. In addition, the mRNA and protein expression levels of GSK-3ß, RANK, RANKL and NF-κB in the renal tissues were detected using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. As compared with the NC group, the level of urinary protein was significantly increased in the DNM group (P<0.05); however, as compared with the DNM Group, the level of urinary protein at 12 weeks was significantly decreased in the DNI group (P<0.05). As compared with the NC group, marked pathological changes were detected, and the mRNA and protein expression levels of GSK-3ß, RANK, RANKL and NF-κB were significantly increased, in the renal tissues of the DNM group. Conversely, pathological alterations in the renal tissues were attenuated, and the mRNA and protein expression levels of GSK-3ß, RANK, RANKL and NF-κB were significantly decreased (P<0.05), in the DNI group, as compared with the DNM group. The results of the present study suggested that GSK-3ß, RANK, RANKL and NF-κB may be crucially involved in the development of DN, and that LiCl may effectively attenuate DN by reducing the expression levels of GSK-3ß, RANK, RANKL and NF-κB.

Potential Role of Glycogen Synthase Kinase-3ß in Regulation of Myocardin Activity in Human Vascular Smooth Muscle Cells.

Glycogen synthase kinase (GSK)-3ß, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK-3ß results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co-activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study, therefore, aimed to examine whether and how inhibition of GSK-3ß regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK-3ß inhibitors AR-A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22), and ACTA2 (SM α-actin). In human SMCs overexpressing myocardin through the T-REx system, treatment with either GSK-3ß inhibitor also inhibited the expression of CNN1, TAGLN1, and ACTA2. These effects of GSK-3ß inhibitors were mimicked by transfection with GSK-3ß siRNA. Notably, both AR-A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR-A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant-negative GSK-3ß mutant in myocardin-overexpressing SMCs reduced the expression of calponin, SM22, and SM α-actin. As expected, overexpression of constitutively active or wild-type GSK-3ß in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK-3ß reduces myocardin transcriptional activity, suggesting a role for GSK-3ß in myocardin transcriptional activity and smooth muscle differentiation.

Opposite roles of myocardin and atrogin-1 in L6 myoblast differentiation.

L6 rat myoblasts undergo differentiation and myotube formation when cultured in medium containing a low-concentration of serum, but the underlying mechanism is not well understood. The role of atrogin-1, an E3 ligase with well-characterized roles in muscle atrophy, has not been defined in muscle differentiation. Myocardin is a coactivator of serum response factor (SRF), which together promotes smooth muscle differentiation. Myocardin is transiently expressed in skeletal muscle progenitor cells with inhibitory effects on the expression of myogenin and muscle differentiation. It remains unknown whether myocardin, which undergoes ubiquitination degradation, plays a role in L6 cell differentiation. The current study aimed to investigate the potential roles of myocardin and atrogin-1 in differentiation of L6 cells. As reported by many others, shifting to medium containing 2% serum induced myotube formation of L6 cells. Differentiation was accompanied by up-regulation of atrogin-1 and down-regulation of myocardin, suggesting that both may be involved in muscle differentiation. As expected, over-expression of atrogin-1 stimulated the expression of troponin T and myogenin and differentiation of the L6 myoblasts. Co-expression of myocardin with atrogin-1 inhibited atrogin-1-induced myogenin expression. Over-expression of atrogin-1 decreased myocardin protein level, albeit without affecting its mRNA level. Small-interfering RNA-mediated knockdown of atrogin-1 increased myocardin protein. Consistently, ectopic expression of myocardin inhibited myogenic differentiation. Unexpectedly, myocardin decreased the expression of atrogin-1 without involving Foxo1. Taken together, our results have demonstrated that atrogin-1 plays a positive role in skeletal muscle differentiation through down-regulation of myocardin.

Corn baked by burning coal triggered overexpression of osteopontin in hepatocytes of rats following fluorosis.

OBJECTIVE: To observe the expression of osteopontin (OPN) in hepatocytes of rats fed with corn baked by burning coal from fluorosis areas and a deficiency of calcium/protein intake following fluorosis. METHODS: A total of 48 Wistar rats as objects were randomly assorted into four groups: dose-free fluorine group, which were mainly fed with fluorine-free corn (56% structurally), dose-free fluorine with biased dietary group, which were fed with lower contents of protein (119.41 g/kg) and calcium (0.68 g/kg), high-dose fluorine group (fluorine contents: 104.2 mg/kg), and high-dose fluorine with biased dietary group. After 180 days of cultivation, the contents of fluorine in the bones of rats were tested for the assessment of construction of fluorosis animal model. And the expression of OPN in hepatocytes of rats in different groups was detected with immunohistochemistry and reverse transcription polymerase chain reaction. RESULTS: The present study validated the result that OPN was overexpressed in hepatocytes following fluorosis after oral intake of burning coal-baked corn. OPN was expressed most significantly in high fluorine with biased dietary group, and the high-fluorine group ranked the second most; and dose-free fluorine with biased dietary group ranked the third. The dose-free fluorine group expressed the least OPN. CONCLUSION: Overexpression of OPN in hepatocytes following fluorosis after excess fluorine intake was involved in liver damage process, which was enhanced by deficiency of calcium and protein intake. The results also demonstrated that the development of fluorosis in Guizhou province was correlated with local baking staple corn as a way of excess intake of fluorine and deficiency of calcium/protein intake.