The fusion of monocyte/macrophage lineage cells into fully active, multinucleated, bone resorbing osteoclasts is a complex cell biological phenomenon that utilizes specialized proteins. OC-STAMP, a multi-pass transmembrane protein, has been shown to be required for pre-osteoclast fusion and for optimal bone resorption activity. A previously reported knockout mouse model had only mononuclear osteoclasts with markedly reduced resorption activity in vitro, but with paradoxically normal skeletal micro-CT parameters. To further explore this and related questions, we used mouse ES cells carrying a gene trap allele to generate a second OC-STAMP null mouse strain. Bone histology showed overall normal bone form with large numbers of TRAP-positive, mononuclear osteoclasts. Micro-CT parameters were not significantly different between knockout and wild type mice at 2 or 6 weeks old. At 6 weeks, metaphyseal TRAP-positive areas were lower and mean size of the areas were smaller in knockout femora, but bone turnover markers in serum were normal. Bone marrow mononuclear cells became TRAP-positive when cultured with CSF-1 and RANKL, but they did not fuse. Expression levels of other osteoclast markers, such as cathepsin K, carbonic anhydrase II, and NFATc1, were not significantly different compared to wild type. Actin rings were present, but small, and pit assays showed a 3.5-fold decrease in area resorbed. Restoring OC-STAMP in knockout cells by lentiviral transduction rescued fusion and resorption. N- and C-termini of OC-STAMP were intracellular, and a predicted glycosylation site was shown to be utilized and to lie on an extracellular loop. The site is conserved in all terrestrial vertebrates and appears to be required for protein stability, but not for fusion. Based on this and other results, we present a topological model of OC-STAMP as a 6-transmembrane domain protein. We also contrast the osteoclast-specific roles of OC- and DC-STAMP with more generalized cell fusion mechanisms.
Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1–3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions.
Three named cell types degrade and remove skeletal tissues during growth, repair, or disease: osteoclasts, chondroclasts, and septoclasts. A fourth type, unnamed and less understood, removes non-mineralized cartilage during development of secondary ossification centers. “Osteoclasts,” best known and studied, are polykaryons formed by fusion of monocyte precursors under the influence of CSF-1 (M-CSF) and RANKL. They resorb bone during growth, remodeling, repair, and disease. “Chondroclasts”, originally described as highly similar in cytological detail to osteoclasts, reside on and degrade mineralized cartilage. They may be identical to osteoclasts, since to date there are no distinguishing markers for them. Because osteoclasts also consume cartilage cores along with bone during growth, the term “chondroclast” might best be reserved for cells attached only to cartilage. “Septoclasts” are less studied and appreciated. They are mononuclear perivascular cells rich in cathepsin B. They extend a cytoplasmic projection with a ruffled membrane and degrade the last transverse septum of hypertrophic cartilage in the growth plate, permitting capillaries to bud into it. To do this, antiangiogenic signals in cartilage must give way to vascular trophic factors, mainly VEGF. The final cell type excavates cartilage canals for vascular invasion of articular cartilage during development of secondary ossification centers. The “clasts” are considered in the context of fracture repair and diseases such as arthritis and tumor metastasis. Many observations support an essential role for hypertrophic chondrocytes in recruiting septoclasts and osteoclasts/chondroclasts by supplying VEGF and RANKL. The intimate relationship between blood vessels and skeletal turnover and repair is also examined.
Mammalian MBNL (muscleblind-like) proteins are regulators of alternative splicing and have been implicated in myotonic dystrophy, the most common form of adult onset muscular dystrophy. MBNL3 functions as an inhibitor of muscle differentiation and is expressed in proliferating muscle precursor cells but not in differentiated skeletal muscle. Here we demonstrate that MBNL3 regulates the splicing pattern of the muscle transcription factor myocyte enhancer factor 2 (Mef2) by promoting exclusion of the alternatively spliced -exon. Expression of the transcriptionally more active (؉) isoform of Mef2D was sufficient to overcome the inhibitory effects of MBNL3 on muscle differentiation. These data suggest that MBNL3 antagonizes muscle differentiation by disrupting Mef2 -exon splicing. MBNL3 regulates Mef2D splicing by directly binding to intron 7 downstream of the alternatively spliced exon in the pre-mRNA. The RNA binding activity of MBNL3 requires the CX 7 CX 4 -6 CX 3 H zinc finger domains. Using a cell culture model of myotonic dystrophy and myotonic dystrophy patient tissue, we have evidence that expression of CUG expanded RNAs can lead to an increase in MBNL3 expression and a decrease in Mef2D -exon splicing. These studies suggest that elevating MBNL3 activity in myogenic cells could lead to muscle degeneration disorders such as myotonic dystrophy.
Skeletal muscle regeneration is mediated by myoblasts that undergo epigenomic changes to establish the gene expression program of differentiated myofibers. mSWI/SNF chromatin remodeling enzymes coordinate with lineage-determining transcription factors to establish the epigenome of differentiated myofibers. Bromodomains bind to acetylated lysines on histone N-terminal tails and other proteins. The mutually exclusive ATPases of mSWI/SNF complexes, BRG1 and BRM, contain bromodomains with undefined functional importance in skeletal muscle differentiation. Pharmacological inhibition of mSWI/SNF bromodomain function using the small molecule PFI-3 reduced differentiation in cell culture and in vivo through decreased myogenic gene expression, while increasing cell cycle-related gene expression and the number of cells remaining in the cell cycle. Comparative gene expression analysis with data from myoblasts depleted of BRG1 or BRM showed that bromodomain function was required for a subset of BRG1- and BRM-dependent gene expression. Reduced binding of BRG1 and BRM after PFI-3 treatment showed that the bromodomain is required for stable chromatin binding at target gene promoters to alter gene expression. Our findings demonstrate that mSWI/SNF ATPase bromodomains permit stable binding of the mSWI/SNF ATPases to promoters required for cell cycle exit and establishment of muscle-specific gene expression.
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