Osteoclasts are specialized macrophages that resorb bone. Mice lacking the AP-1 component c-Fos are osteopetrotic because of a lack of osteoclast differentiation and show an increased number of macrophages. The nature of the critical function of c-Fos in osteoclast differentiation is not known. Microarray analysis revealed that Nfatc1, another key regulator of osteoclastogenesis, was down-regulated in Fos^-/- osteoclast precursors. Chromatin immunoprecipitation assay showed that c-Fos bound to the Nfatc1 and Acp5 promoters in osteoclasts. In vitro promoter analyses identified nuclear factor of activated T-cells (NFAT)/AP-1 sites in the osteoclast-specific Acp5 and Calcr promoters. Moreover, in Fos^-/- precursors gene transfer of an active form of NFAT restored transcription of osteoclast-specific genes in the presence of receptor activator of the NF-κB ligand (RANKL), rescuing bone resorption. In the absence of RANKL, however, Fos^-/- precursors were insensitive to NFAT-induced osteoclastogenesis unlike wild-type precursors. These data indicate that lack of Nfatc1 expression is the cause of the differentiation block in Fos^-/- osteoclast precursors and that transcriptional induction of Nfatc1 is a major function of c-Fos in osteoclast differentiation.

Amylin is a member of the calcitonin family of hormones cosecreted with insulin by pancreatic β cells. Cell culture assays suggest that amylin could affect bone formation and bone resorption, this latter function after its binding to the calcitonin receptor (CALCR). Here we show that Amylin inactivation leads to a low bone mass due to an increase in bone resorption, whereas bone formation is unaffected. In vitro, amylin inhibits fusion of mononucleated osteoclast precursors into multinucleated osteoclasts in an ERK1/2-dependent manner. Although Amylin +/− mice like Amylin-deficient mice display a low bone mass phenotype and increased bone resorption, Calcr +/− mice display a high bone mass due to an increase in bone formation. Moreover, compound heterozygote mice for Calcr and Amylin inactivation displayed bone abnormalities observed in both Calcr +/− and Amylin +/− mice, thereby ruling out that amylin uses CALCR to inhibit osteoclastogenesis in vivo. Thus, amylin is a physiological regulator of bone resorption that acts through an unidentified receptor.

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 39 enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role.

The signals that control skeletogenesis are incompletely understood. Here we show that deleting Kindlin-2 in Prx1-expressing mesenchymal progenitors in mice causes neonatal lethality, chondrodysplasia and loss of the skull vault. Kindlin-2 ablation reduces chondrocyte density by decreasing cell proliferation and increasing apoptosis, and disrupts column formation, thus impairing the formation of the primary ossification center and causing severe limb shortening. Remarkably, Kindlin-2 localizes to not only focal adhesions, but also to the nuclei of chondrocytes. Loss of Kindlin-2 reduces, while the overexpression of Kindlin-2 increases, Sox9 expression. Furthermore, the overexpression of Sox9 restores the defects in chondrogenic differentiation induced by Kindlin-2 deletion in vitro. In addition, Kindlin-2 ablation inhibits TGF-β1-induced Smad2 phosphorylation and chondrocyte differentiation. Finally, deleting Kindlin-2 in chondrocytes directly impairs chondrocyte functions, resulting in progressive dwarfism and kyphosis in mice. These studies uncover a previously unrecognized function for Kindlin-2 and a mechanism for regulation of the chondrocyte differentiation programme and chondrogenesis.

Galson DL, et al. J Bone Metab. 2014 May;21(2):85-98. http://dx.doi.org/10.11005/jbm.2014.21.2.85