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In a previous transcriptomic study of human bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells"), SFRP2 was highly over-represented in a subset of multipotent BMSCs (skeletal stem cells, SSCs), which recreate a bone/marrow organ in an in vivo ectopic bone formation assay. SFRPs modulate WNT signaling, which is essential to maintain skeletal homeostasis, but the specific role of SFRP2 in BMSCs/SSCs is unclear. Here, we evaluated Sfrp2 deficiency on BMSC/SSC function in models of skeletal organogenesis and regeneration. The skeleton of Sfrp2-deficient (KO) mice is overtly normal; but their BMSCs/SSCs exhibit reduced colony-forming efficiency, reflecting low SSC self-renewal/abundancy. Sfrp2 KO BMSCs/SSCs formed less trabecular bone than those from WT littermates in the ectopic bone formation assay. Moreover, regeneration of a cortical drilled hole defect was dramatically impaired in Sfrp2 KO mice. Sfrp2-deficient BMSCs/SSCs exhibited poor in vitro osteogenic differentiation as measured by Runx2 and Osterix expression and calcium accumulation. Interestingly, activation of the Wnt co-receptor, Lrp6, and expression of Wnt target genes, Axin2, C-myc and Cyclin D1, were reduced in Sfrp2-deficient BMSCs/SSCs. Addition of recombinant Sfrp2 restored most of these activities, suggesting that Sfrp2 acts as a Wnt agonist. We demonstrate that Sfrp2 plays a role in self-renewal of SSCs and in the recruitment and differentiation of adult SSCs during bone healing. SFRP2 is also a useful marker of BMSC/SSC multipotency, and a factor to potentially improve the quality of ex vivo expanded BMSC/SSC products. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported. Therefore, to generate lineage‐specific OPs from human embryonic stem cells and human induced pluripotent stem cells, we employed stepwise differentiation of paraxial mesoderm‐like cells, lateral plate mesoderm‐like cells, and neural crest‐like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. We also report, for the first time, high FGF1 levels in neural crest‐derived OPs—a notable finding given the critical role of fibroblast growth factors (FGFs) in osteogenesis and mineral homeostasis. Our results indicate that FGF1 influences RUNX2 levels, with concomitant changes in ERK1/2 signaling. Overall, our study further validates hPSCs' power to model bone development and disease and reveals new, potentially important pathways influencing these processes. [ABSTRACT FROM AUTHOR]

Fibrous dysplasia (FD) is a skeletal disease caused by somatic activating mutations of the cyclic adenosine monophosphate (cAMP)-regulating protein, α-subunit of the Gs stimulatory protein (Gsα). These mutations lead to replacement of normal bone by proliferative osteogenic precursors, resulting in deformity, fracture, and pain. Medical treatment has been ineffective in altering the disease course. Receptor activator of NF-κB ligand (RANKL) is a cell-surface protein involved in many cellular processes, including osteoclastogenesis, and is reported to be overexpressed in FD-like bone cells. Denosumab is a humanized monoclonal antibody to RANKL approved for treatment of osteoporosis and prevention of skeletal-related events from bone metastases. We present the case of a 9-year-old boy with severe FD who was treated with denosumab for a rapidly expanding femoral lesion. Immunohistochemical staining on a pretreatment bone biopsy specimen revealed marked RANKL expression. He was started on monthly denosumab, with an initial starting dose of 1 mg/kg and planned 0.25 mg/kg dose escalations every 3 months. Over 7 months of treatment he showed marked reduction in pain, bone turnover markers (BTMs), and tumor growth rate. Denosumab did not appear to impair healing of a femoral fracture that occurred while on treatment. With initiation of treatment he developed hypophosphatemia and secondary hyperparathyroidism, necessitating supplementation with phosphorus, calcium, and calcitriol. BTMs showed rapid and sustained suppression. With discontinuation there was rapid and dramatic rebound of BTMs with cross-linked C-telopeptide (reflecting osteoclast activity) exceeding pretreatment levels, accompanied by severe hypercalcemia. In this child, denosumab lead to dramatic reduction of FD expansion and FD-related bone pain. Denosumab was associated with clinically significant disturbances of mineral metabolism both while on treatment and after discontinuation. Denosumab treatment of FD warrants further study to confirm efficacy and determine potential morbidity, as well as to determine the mechanism of RANKL in the pathogenesis of FD and related bone marrow stromal cell diseases. © 2012 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor-23 (FGF23) is a phosphate- and vitamin D-regulating hormone derived from osteoblasts/osteocytes that circulates in both active (intact, iFGF23) and inactive (C-terminal, cFGF23) forms. O-glycosylation by O-glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3) and differential cleavage by furin have been shown to be involved in regulating the ratio of active to inactive FGF23. Elevated iFGF23 levels are observed in a number of hypophosphatemic disorders, such as X-linked, autosomal recessive, and autosomal dominant hypophosphatemic rickets, whereas low iFGF23 levels are found in the hyperphosphatemic disorder familial tumoral calcinosis/hyperphosphatemic hyperostosis syndrome. Fibrous dysplasia of bone (FD) is associated with increased total FGF23 levels (cFGF23 + iFGF23); however, classic hypophosphatemic rickets is uncommon. Our results suggest that it can be explained by increased FGF23 cleavage leading to an increase in inactive cFGF23 relative to active iFGF23. Given the fact that FD is caused by activating mutations in the small G-protein Gsα that results in increased cyclic adenosine monophosphate (cAMP) levels, we postulated that there may be altered FGF23 cleavage in FD and that the mechanism may involve alterations in cAMP levels and ppGalNacT3 and furin activities. Analysis of blood specimens from patients with FD confirmed that the elevated total FGF23 levels are the result of proportionally increased cFGF23 levels, consistent with less glycosylation and enhanced cleavage by furin. Analysis of primary cell lines of normal and mutation-harboring bone marrow stromal cells (BMSCs) from patients with FD demonstrated that BMSCs harboring the causative Gsα mutation had higher cAMP levels, lower ppGalNAcT3, and higher furin activity. These data support the model wherein glycosylation by ppGalNAcT3 inhibits FGF23 cleavage by furin and suggest that FGF23 processing is a regulated process that controls overall FGF23 activity in FD patients. © 2012 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

The btomodomain protein. BRD4, has been identified recently as a therapeusic target in acute myeloid leukemia, multiple myeloma, Burkitt's lymphoma. NUT midline carcinoma, colon cancer, and inflammatory disease; its loss is a prognostic signature for meta- static breast cancer. BRD4 also contributes to regulation of both cell cycle and transcription of oncogenes. HIV, and human papil- loma virus (HPV). Despite its role in a broad range of biological processes, the precise molecular mechanism of BRD4 function remains unknown. We report that BRD4 is an atypical kinase that binds to the carboxyl-terminal domain (GD) of RNA polymerase II and directly phosphorylates its serine 2 (Ser2) sites both in vitro and in vivo under conditions where other GD kinases are inactive. Phosphorylation of the GD Ser2 is inhibited in vivo by a BRD4 inhibitor that blocks its binding to chromatin. Our finding that BRD4 is an RNA polymerase II GD Ser2 kinase implicates it as a regulator of eukaryotic transcription. [ABSTRACT FROM AUTHOR]

Skeletal dysplasias are common disabling disorders characterized by aberrant growth of bone and cartilage leading to abnormal skeletal structures and functions, often attributable to defects in skeletal progenitor cells. The underlying molecular and cellular mechanisms of most skeletal dysplasias remain elusive. Although the Wnt/β-catenin signaling pathway is required for skeletal progenitor cells to differentiate along the osteoblastic lineage, inappropriately elevated levels of signaling can also inhibit bone formation by suppressing osteoblast maturation. Here, we investigate interactions of the four major Gα protein families (Gαs, Gαi/o, Gαq/11, and Gα12/13) with the Wnt/β-catenin signaling pathway and identify a causative role of Wnt/β-catenin signaling in fibrous dysplasia (FD) of bone, a disease that exhibits abnormal differentiation of skeletal progenitor cells. The activating Gαs mutations that cause FD potentiated Wnt/β-catenin signaling, and removal of Gαs led to reduced Wnt/β-catenin signaling and decreased bone formation. We further show that activation of Wnt/β-catenin signaling in osteoblast progenitors results in an FD-like phenotype and reduction of β-catenin levels rescued differentiation defects of FD patient-derived stromal cells. Gα proteins may act at the level of β-catenin destruction complex assembly by binding Axin. Our results indicate that activated Gα proteins differentially regulate Wnt/β-catenin signaling but, importantly, are not required core components of Wnt/β-catenin signaling. Our data suggest that activated Gα proteins are playing physiologically significant roles during both skeletal development and disease by modulating Wnt/β-catenin signaling strength. [ABSTRACT FROM AUTHOR]

The article reports on the results of research which was conducted in an effort to determine the role that somatic mosaicism in fibrous dysplasia of bone within the context of skeletal (mesenchymal) stem cells play. Researchers assessed the frequency of mutated colony forming unit-fibroblasts from lesions and other sites in patients. They found that as lesions age, mutant stem cells survive, self renew and enable formation of a normal structure and that GNAS mutations disrupt cell renewal.

The article presents the clinical, histological, and genetic studies of two cases of isolated fibro-osseous lesions of the femur in adults. It shows the overlap between monostotic fibrous dysplasia (MFD) of the proximal femur and the liposclerosin myxofibrous tumor. The two cases highlight how the incomplete understanding of the natural history of MFD may result in diagnostic pitfalls or incorrect classification of individual lesions.

G protein–coupled receptors (GPCRs) mediate effects of extracellular signaling molecules in all the body's cells. These receptors are encoded by scarce mRNAs; therefore, detecting their transcripts with conventional microarrays is difficult. We present a method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression with as little as 1 μg of total RNA, and using it, we profiled three human bone marrow stromal cell (BMSC) lines. [ABSTRACT FROM AUTHOR]

Somatic gain‐of‐function mutations of GNAS cause a spectrum of clinical phenotypes, ranging from McCune‐Albright syndrome (MAS) to isolated disease of bone, endocrine glands, and more rarely, other organs. In MAS, a syndrome classically characterized by polyostotic fibrous dysplasia (FD), café‐au‐lait (CAL) skin spots, and precocious puberty, the heterogenity of organ involvement, age of onset, and clinical severity of the disease are thought to reflect the variable size and the random distribution of the mutated cell clone arising from the postzygotic mutation. We report a case of neonatal MAS with hypercortisolism and cholestatic hepatobiliary dysfunction in which bone changes indirectly emanating from the disease genotype, and distinct from FD, led to a fatal outcome. Pulmonary embolism of marrow and bone fragments secondary to rib fractures was the immediate cause of death. Ribs, and all other skeletal segments, were free of changes of typical FD and fractures appeared to be the result of a mild‐to‐moderate degree of osteopenia. The mutated allele was abundant in the adrenal glands and liver, but not in skin, muscle, and fractured ribs, where it could only be demonstrated using a much more sensitive PNA hybridization probe‐based FRET (Förster resonance energy transfer) technique. Histologically, bilateral adrenal hyperplasia and cholestatic disease matched the abundant disease genotype in the adrenals and liver. Based on this case and other sporadic reports, it appears that gain‐of‐function mutations of GNAS underlie a unique syndromic profile in neonates characterized by CAL skin spots, hypercortisolism, hyperthyroidism, hepatic and cardiac dysfunction, and an absence (or latency) of FD, often with a lethal outcome. Taken together, our and previous cases highlight the phenotypic severity and the diagnostic and therapeutic challenges of MAS in neonates. Furthermore, our case specifically points out how secondary bone changes, unrelated to the direct impact of the mutation, may contribute to the unfavorable outcome of very early‐onset MAS. © 2018 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]