Hypoxia and inflammation are implicated in the episodic induction of heterotopic endochondral ossification (HEO); however, the molecular mechanisms are unknown. HIF-1α integrates the cellular response to both hypoxia and inflammation and is a prime candidate for regulating HEO. We investigated the role of hypoxia and HIF-1α in fibrodysplasia ossificans progressiva (FOP), the most catastrophic form of HEO in humans. We found that HIF-1α increases the intensity and duration of canonical bone morphogenetic protein (BMP) signaling through Rabaptin 5 (RABEP1)-mediated retention of Activin A receptor, type I (ACVR1), a BMP receptor, in the endosomal compartment of hypoxic connective tissue progenitor cells from patients with FOP. We further show that early inflammatory FOP lesions in humans and in a mouse model are markedly hypoxic, and inhibition of HIF-1α by genetic or pharmacologic means restores canonical BMP signaling to normoxic levels in human FOP cells and profoundly reduces HEO in a constitutively active Acvr1Q207D/+ mouse model of FOP. Thus, an inflammation and cellular oxygen-sensing mechanism that modulates intracellular retention of a mutant BMP receptor determines, in part, its pathologic activity in FOP. Our study provides critical insight into a previously unrecognized role of HIF-1α in the hypoxic amplification of BMP signaling and in the episodic induction of HEO in FOP, and further identifies HIF-1α as a therapeutic target for FOP and perhaps non-genetic forms of HEO.

Cells with osteogenic potential can be found in a variety of tissues. Here we show that circulating osteogenic precursor (COP) cells, a bone marrow-derived type I collagen+/CD45+ subpopulation of mononuclear adherent cells, are present in early pre-osseous fibroproliferative lesions in patients with fibrodysplasia ossificans progressiva (FOP) and nucleate heterotopic ossification (HO) in a murine in vivo implantation assay. Blood samples from FOP patients with active episodes of HO contain significantly higher numbers of clonally-derived COP cell colonies than patients with stable disease or unaffected individuals. The highest level of COP cells was found in a patient just prior to the clinical onset of an HO exacerbation. Our studies show that even COP cells derived from an unaffected individual can contribute to HO in genetically susceptible host tissue. The possibility that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites has tremendous implications and, to our knowledge, represents the first example of their involvement in clinical HO. Thus, bone formation is not limited to cells of the mesenchymal lineage, and circulating cells of hematopoietic origin can also serve as osteogenic precursors at remote sites of tissue inflammation.

The E2A gene encodes two basic helix-loop-helix proteins designated E12 and E47. Although these proteins are widely expressed, they are required only for the B-lymphocyte lineage where DNA binding is mediated distinctively by E47 homodimers. By studying the properties of deltaE47, an N-terminal truncation of E47, we provide evidence that phosphorylation may contribute to B-cell-specific DNA binding by E47. Two serines N terminal to the deltaE47 basic helix-loop-helix domain were found to be phosphorylated in a variety of cell types but were hypophosphorylated in B cells. Phosphorylating these serines in vitro inhibited DNA binding by deltaE47 homodimers but not by deltaE47-containing heterodimers, such as deltaE47:MyoD. These results argue that hypophosphorylation may be a prerequisite for activity of E47 homodimers in B cells, suggesting the use of an inductive (nonstochastic) step in early B-cell development.

Early B-cell factor (EBF) is a DNA binding protein required for early B-cell development. It activates transcription of several B-cell-specific genes, including the 5 gene, which encodes a protein necessary for signaling by the pre-B-cell receptor. In an effort to understand the mechanism by which EBF activates transcription, we examined its interaction with the coactivator protein p300/CBP. We found that two domains of EBF each bind the histone acetyltransferase (HAT)/CH3 domain of p300/CBP both in vitro and in vivo. Surprisingly, transcriptional activation by EBF was not sensitive to E1A, a potent p300/CBP inhibitor. In fact, overexpressed EBF mimicked E1A by severely repressing the activity of several other transcription factors, including E47, a protein that acts cooperatively with EBF to promote transcription of the 5 gene. This broad inhibitory profile correlated with EBF's ability to repress the HAT activity of p300/CBP in vivo and in vitro. However, such a repressed complex is not likely to form at the 5 promoter in vivo since (i) EBF could not bind p300/CBP and DNA simultaneously and (ii) the cooperativity imparted by E47 was sensitive to E1A. Our data reveal an intriguing inhibitory property of EBF-a property shared only by E1A, Twist, Pu.1, and the Hox family of homeodomain proteins-and suggest that E47 and EBF play distinct roles during 5 promoter activation.