1,25(OH)2vitamin D3 enhances myogenic differentiation by modulating the expression of key angiogenic growth factors and angiogenic inhibitors in C2C12 skeletal muscle cells

Abstract

Vitamin D is mostly recognized for its regulation of calcium homeostasis in relation to the intestine, kidney, and bone. Although clinical studies have linked vitamin D with increased muscle function and strength, little is known of its underlying molecular mechanism. We recently demonstrated that 1,25-D3 exerts a direct pro-myogenic effect on skeletal muscle cells; this has provoked our investigation of 1,25-D’s effect on angiogenesis, a vital process for new capillary development and tissue repair. In this study, we examined the mechanism by which 1,25-D3 modulates key angiogenic growth factors and angiogenic inhibitors. C2C12 myoblasts were incubated with 100 nM 1,25-D3 or placebo for 1, 4 and 10 days. At the end of the respective incubation time, total RNA was isolated for PCR arrays and for qRT-PCR. Total proteins were isolated for western blots and proteome profiler arrays. The addition of 1,25-D3 to C2C12 myoblasts increased VEGFa and FGF-1: two pro-angiogenic growth factors that promote neo-vascularization and tissue regeneration, and decreased FGF-2 and TIMP-3: two myogenic and/or angiogenic inhibitors. Our previous study demonstrated that 1,25-D3 altered IGF-I/II expression, consistent with the observed changes in VEGFa and FGF-2 expression. These results extend our previous findings and demonstrate the modulation of angiogenesis which may be an additional mechanism by which 1,25-D3 promotes myogenesis. This study supports the mechanistic rationale for assessing the administration of vitamin D and/or vitamin D analogues to treat select muscle disorders and may also provide an alternative solution for therapies that directly manipulate VEGF and FGF’s to promote angiogenesis.

1,25-vitamin D3 promotes cardiac differentiation through modulation of the WNT signaling pathway

“…The 100nM supra-physiological concentration of 1,25-D 3 applied in the experimental design was based on the present and previous dose-response studies and it is in alignment with a commonly used dose on different cell lines or in primary cell culture studies (Barbosa et al 2004, Cardus et al 2006, Artaza et al 2010, Khanna-jain et al 2010, Ramirez et al 2010. Because of the short half-life of 1,25-D 3 , the cell culture media was replaced every 24h (Garcia et al 2011(Garcia et al , 2013.…”

“…Real time PCRs were performed as follows: melting for 10 min at 95°C, 40 cycles of two-step PCR including melting for 15 sec at 95°C, annealing for 1 min at 60°C. The raw data were analyzed using the ΔΔC t method following the manufacturer's instructions (SABiosciences Corp., Frederick, MD, USA) (Garcia et al 2011(Garcia et al , 2013.…”

“…Rat gene PCR primer sets (RT2) for Mki67, PCNA, Casp3, Bcl-2, Casein kinase 1 α-1 and Wnt11 were obtained from SABiosciences Corp (Frederick, MD, USA). The QIAGEN Sybr Green PCR kit with HotStar Taq DNA polymerase (QIAGEN, Valencia, CA, USA) was used with i-Cycler PCR thermocycler and fluorescent detector lid (Bio-Rad, Hercules, CA, USA) (Garcia et al 2011(Garcia et al , 2013). …”

“…Samples of 25 ng cDNA were analyzed in quadruplicate in parallel with GAPDH controls; standard curves (threshold cycle vs. log pg cDNA) were generated by log dilutions of from 0.1 pg to 100 ng standard cDNA (reverse-transcribed mRNA from H9c2 cells in AM). Experimental mRNA starting quantities were then calculated from the standard curves and averaged using i-Cycler, iQ software as described previously (Garcia et al, 2013;. The ratios of marker experimental gene (e.g.…”

“…After several washes, the immunoreactive bands were visualized using the SuperSignal western blotting chemiluminescence detection system (Thermo Fisher Scientific, Inc., Rockford, IL, USA). The densitometry analysis of the bands was done with the Scion Image software beta 4.02 (Scion Corp., Frederick, MD, USA) (Garcia et al 2011(Garcia et al , 2013.…”