Serum induction of the collagen X promoter requires the Raf/MEK/ERK and p38 pathways.

The collagen X gene is expressed exclusively by differentiated, hypertrophic chondrocytes. The mechanisms controlling collagen X expression remain largely unknown. Here we show that collagen X promoter activity can be induced by serum stimulation of chondrogenic MCT cells. The serum response is conferred by a 462 nucleotide promoter fragment. Both the c-Raf/MEK/ERK and p38 MAP kinase pathways are required for this effect, whereas phosphatidylinositol-3-kinase and protein kinase A repress promoter activation. These data are the first to demonstrate serum inducibility of the collagen X promoter and to identify signal transduction pathways involved.     

FGF2 stimulates the proliferation of human mesenchymal stem cells through the transient activation of JNK signaling

FGF2 has been shown to enhance proliferation and maintain differentiation potential in hMSCs during in vitro propagation. In this study, we investigated the role of mitogen-activated protein kinase in the functions of FGF2 in hMSCs. We demonstrated that FGF2 induces the transient activation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated protein kinase or p38 protein kinase. SP600125 and a dominant negative JNK1 significantly reduced the FGF2-enhanced proliferation of hMSCs. Treatment with SP600125 also diminished the activity of FGF2 in the maintenance of adipogenic and osteogenic differentiation potential. These results suggest that JNK signaling is involved in the FGF2-induced stimulation of the proliferation and the maintenance of differentiation potential in hMSCs.     

B-Myb represses trans-activation of the Col5A2 collagen promoter indirectly via inhibition of binding of factors interacting with positive elements within the first exon.

B-myb, a member of the myb gene family, was originally isolated based on its high homology with c-myb in the DNA-binding domain. Previously we showed that B-myb is expressed in bovine vascular smooth muscle cells (SMCs) in a cell cycle-dependent fashion, and inhibits type I collagen gene promoter activity. Here, we have explored its role in regulation of another fibrillar collagen gene, Col5A2, encoding the (alpha2 chain of type V collagen. Ectopic expression of B-Myb decreased alpha 2(V) promoter activity and endogenous alpha 2(V) collagen mRNA levels. The responsive region of the alpha 2(V) collagen gene was localized to a fragment including 100 bp of basal promoter and 150 bp of exon 1 sequences, which contained two CRE-like elements. Binding to these elements increased upon deprivation of serum-growth factors, when expression of the Col5A2 gene is elevated, leading us to test their role despite the failure of excess unlabelled CRE oligonucleotide from the somatostatin gene to successfully compete for binding. Mutation of the elements significantly decreased the basal level of alpha2(V) collagen promoter activity and ablated inhibition by B-Myb. Furthermore, addition of B-Myb-glutathionine S-transferase fusion protein inhibited complex formation. Thus, these results confirm a major role for B-Myb in mediating intracellular signals controlling collagen gene expression in vascular SMCs. A model of indirect repression of the Col5A2 gene by B-Myb, via interaction with a positively-acting matrix regulatory factor, termed MRF-V, is discussed.     

Psoralen stimulates osteoblast differentiation through activation of BMP signaling

Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. In order to improve the treatment of osteoporosis, identification of anabolic and orally available agents with minimal side effects is highly desirable. Psoralen is a coumarin-like derivative extracted from Chinese herbs, which have been used to treat bone diseases for thousands of years. However, the role of Psoralen in osteoblast function and the underlying molecular mechanisms remain poorly understood. In this study, we found that Psoralen promoted osteoblast differentiation in primary mouse calvarial osteoblasts in a dose-dependent manner, demonstrated by up-regulation of expressions of osteoblast-specific marker genes including type I collagen, osteocalcin and bone sialoprotein and enhancement of alkaline phosphatase activity. We further demonstrated that Psoralen up-regulated the expression of Bmp2 and Bmp4 genes, increased the protein level of phospho-Smad1/5/8, and activated BMP reporter (12xSBE-OC-Luc) activity in a dose-dependent manner, as well as enhanced the expression of Osx, the direct target gene of BMP signaling. Deletion of the Bmp2 and Bmp4 genes abolished the stimulatory effect of Psoralen on the expression of osteoblast marker genes, such as Col1, Alp, Oc and Bsp. Our results suggest that Psoralen acts through the activation of BMP signaling to promote osteoblast differentiation and demonstrate that Psoralen could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.