Scleraxis and NFATc regulate the expression of the pro-alpha1(I) collagen gene in tendon fibroblasts.

The combinatorial action of separate cis-acting elements controls the cell-specific expression of type I collagen genes. In particular, we have shown that two short elements located between -3.2 and -2.3 kb and named TSE1 and TSE2 are needed for expression of the mouse COL1a1 gene in tendon fibroblasts. In this study, we analyzed the trans-acting factors binding to TSE1 and TSE2. Gel shift experiments showed that scleraxis (SCX), which is a basic helix-loop-helix transcription factor that is expressed selectively in tendon fibroblasts, binds TSE2, preferentially as a SCX/E47 heterodimer. In transfection experiments, overexpression of SCX and E47 strongly enhanced the activity of reporter constructs harboring either four copies of TSE2 cloned upstream of the COL1a1 minimal promoter or a 3.2-kb segment of the COL1a1 proximal promoter. Analysis of TSE1 showed that it contains a consensus binding site for NFATc transcription factors. This led us to show that the NFATc4 gene is expressed in tendons of developing mouse limbs and in TT-D6 cells, a cell line that has characteristics of tendon fibroblasts. In gel shift assays, TSE1 bound NFATc proteins present in nuclear extracts from TT-D6 cells. In transfection experiments, overexpression of NFATc transactivated a reporter construct harboring four copies of TSE1 cloned upstream of the COL1a1 minimal promoter. By contrast, inhibition of the nuclear translocation of NFATc proteins in TT-D6 cells strongly inhibited the expression of the COL1a1 gene. Taken together, these results suggest that SCX and NFATc4 cooperate to activate the COL1a1 gene specifically in tendon fibroblasts.

Runx2 is a target of mechanical unloading to alter osteoblastic activity and bone formation in vivo.

Molecular mechanisms underlying unloading-induced reduction of bone formation have not yet been fully understood. In vitro, Runx2 has been suggested to be involved in mechanical signaling in osteoblasts. However, the roles of Runx2 in vivo during the bone response to mechanical stimuli have not yet been known. The purpose of this paper was to examine the roles of Runx2 in unloading-induced bone loss in vivo. Tail suspension was conducted for 2 wk using 9- to 11-wk-old Runx2 heterozygous knockout mice (Runx2(+/-)) and wild-type (Wt) littermates. Bones were subjected to two-dimensional micro-x-ray computed tomography, bone histomorphometry and RT-PCR analyses. Loss of half Runx2 gene dosage-exacerbated unloading-induced bone loss in trabecular and cortical envelopes. Unloading-induced reduction in mineral apposition rate and bone formation rate in cortical bone as well as trabecular bone was exacerbated in Runx2(+/-) mice, compared with Wt mice. Bone resorption parameters were not significantly affected by unloading or Runx2(+/-) genotype. Basal Runx2 and osterix mRNA levels in bone were reduced by 50% in Wt, whereas unloading in Runx2(+/-) mice did not further alter Runx2 and osterix mRNA levels. In contrast, osteocalcin mRNA levels were reduced by unloading, regardless of Runx2 gene dosage. These data demonstrated that full Runx2 gene dosage is required for maintaining normal function of osteoblasts in mechanical unloading or nonphysiological condition. Finally, we propose Runx2 as a critical target gene in unloading to alter osteoblastic activity and bone formation in vivo.