Bioinformatic identification of connective tissue growth factor as an osteogenic protein within skeletal muscle

Abstract

Aging is associated with increasing incidence of osteoporosis; a skeletal disorder characterized by compromised bone strength that may predispose patients to an increased risk of fracture. It is imperative to identify novel ways in which to attenuate such declines in the functional properties of bone. The purpose of this study was to identify, through in silico, in vitro, and in vivo approaches, a protein secreted from skeletal muscle that is putatively involved in bone formation. We performed a functional annotation bioinformatic analysis of human skeletal muscle‐derived secretomes (n = 319) using DAVID software. Cross‐referencing was conducted using OMIM, Unigene, UniProt, GEO, and CGAP databases. Signal peptides and transmembrane residues were analyzed using SignalP and TMHMM software. To further investigate functionality of the identified protein, L6 and C2C12 myotubes were grown for in vitro analysis. C2C12 myotubes were subjected to 16 h of glucose deprivation (GD) prior to analysis. In vivo experiments included analysis of 6‐week calorie restricted (CR) rat muscle samples. Bioinformatic analysis yielded 15 genes of interest. GEO dataset analysis identified BMP5, COL1A2, CTGF, MGP, MMP2, and SPARC as potential targets for further processing. Following TMHMM and SignalP processing, CTGF was chosen as a candidate gene. CTGF expression level was increased during L6 myoblast differentiation (P <0.01). C2C12 myotubes showed no change in response to GD. Rat soleus muscle samples exhibited an increase in CTGF expression (n = 16) in response to CR (35%) (P <0.05). CTGF was identified as a skeletal muscle expressed protein through bioinformatic analysis of skeletal muscle‐derived secretomes and in vitro/in vivo analysis. Future study is needed to determine the role of muscle‐derived CTGF in bone formation and remodeling processes.

Primary familial brain calcification with a novel SLC20A2 mutation: Analysis of PiT‐2 expression and localization

“…The duplication was confirmed and precisely defined by a sub‐cloning experiment (Figure d). The mutation was further analyzed with three on‐line tools (PSIPRED, TMHMM2.0 and TMpred) to predict the structure of the mutant PiT‐2 (Buchan, Minneci, Nugent, Bryson, & Jones, ; Forrester et al, ; Kosciolek & Jones, ). According to TMHMM2.0 and TMpred, which predict the number of transmembrane domains, the duplication caused elongation of the last (XII) transmembrane domain.…”

Altered Transcription Factor Expression Responses to Exercise in Insulin Resistance

“…CTGF is a mito-attractant that is involved in cell adhesion and fibrosis in many cell types, including myofibroblasts ( Ramazani et al, 2018 ). CTGF expression rises after muscle contraction and exercise ( Kivela et al, 2007 ; McLean et al, 2015 ) and is part of what appears to be a coordinated transcriptional response in muscle that involves a variety of extracellular matrix and associated genes that is activated by maneuvers that alter insulin sensitivity ( Richardson et al, 2005 ; Bajaj et al, 2007 ; Forrester et al, 2014 ). The lack of change in CTGF expression following exercise in insulin resistant muscle could be a marker of exercise-induced changes that raise insulin sensitivity or could be part of the underlying mechanism.…”

Therapeutic effect of intra‐articular injection of ribbon‐type decoy oligonucleotides for hypoxia inducible factor‐1 on joint contracture in an immobilized knee animal model

“…Thus, we did not evaluate pathological changes in periarticular bone and cartilage. However, suppression of HIF‐1 may have an effect on osteochondral remodeling because VEGF was reported to be an essential molecule in cartilage metabolism and bone formation and also because CTGF was suggested to be related to bone formation and remodeling processes. Although our histological studies showed no obvious pathological changes in periarticular bone and cartilage in the knee joint at least until 2 weeks after knee fixation, detailed and long‐term evaluation of bone/cartilage metabolism is needed in the future.…”