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Osteoblasts play an increasingly recognized role in supporting hematopoietic development and recently have been implicated in the regulation of B lymphopoiesis. Here we demonstrate that the heterotrimeric G protein [alpha] subunit [G.sub.s][alpha] is required in cells of the osteoblast lineage for normal postnatal B lymphocyte production. Deletion of [G.sub.s][alpha] early in the osteoblast lineage results in a 59% decrease in the percentage of B cell precursors in the bone marrow. Analysis of peripheral blood from mutant mice revealed a 67% decrease in the number of circulating B lymphocytes by 10 days of age. Strikingly, other mature hematopoietic lineages are not decreased significantly. Mice lacking [G.sub.s][alpha] in cells of the osteoblast lineage exhibit a reduction in pro-B and pre-B cells. Furthermore, interleukin (IL)-7 expression is attenuated in [G.sub.s][alpha]-deficient osteoblasts, and exogenous IL-7 is able to restore B cell precursor populations in the bone marrow of mutant mice. Finally, the defect in B lymphopoiesis can be rescued by transplantation into a WT microenvironment. These findings confirm that osteoblasts are an important component of the B lymphocyte niche and demonstrate in vivo that [G.sub.s][alpha]-dependent signaling pathways in cells of the osteoblast lineage extrinsically regulate bone marrow B lymphopoiesis, at least partially in an IL-7-dependent manner. B lymphocyte | G protein | osteoblast

Small noncoding RNAs, microRNAs (miRNAs), bind to messenger RNAs through base pairing to suppress gene expression. Despite accumulating evidence that miRNAs play critical roles in various biological processes across diverse organisms, their roles in mammalian skeletal development have not been demonstrated. Here, we show that Dicer, an essential component for biogenesis of miRNAs, is essential for normal skeletal development. Dicer-null growth plates show a progressive reduction in the proliferating pool of chondrocytes, leading to severe skeletal growth defects and premature death of mice. The reduction of proliferating chondrocytes in Dicer-null growth plates is caused by two distinct mechanisms: decreased chondrocyte proliferation and accelerated differentiation into postmitotic hypertrophic chondrocytes. These defects appear to be caused by mechanisms downstream or independent of the Ihh-PTHrP signaling pathway, a pivotal signaling system that regulates chondrocyte proliferation and differentiation. Microarray analysis of Dicer-null chondrocytes showed limited expression changes in miRNA-target genes, suggesting that, in the majority of cases, chondrocytic miRNAs do not directly regulate target RNA abundance. Our results demonstrate the critical role of the Dicer-dependent pathway in the regulation of chondrocyte proliferation and differentiation during skeletal development. microRNA | skeletal development

Bone morphogenetic proteins (BMPs) play important roles at multiple stages of endochondral bone formation. However, the roles of BMP signaling in chondrocytes in vivo are still contentious. In the present study, we overexpressed a constitutively active BMP receptor 1A (caBmpr1a) in chondrocytes by using two systems: caBmpr1a was directly driven by a rat type II collagen promoter in a conventional transgenic system and indirectly driven in a UASGal4 binary system. CaBmpr1a expression caused shortening of the columnar layer of proliferating chondrocytes and up-regulation of maturation markers, suggesting acceleration of differentiation of proliferating chondrocytes toward hypertrophic chondrocytes. In addition to the acceleration of chondrocyte differentiation, conventional transgenic mice showed widening of cartilage elements and morphological alteration of perichondrial cells, possibly due to stimulation of differentiation of prechondrogenic cells. Moreover, bigenic expression of caBmpr1a rescued the differentiation defect of prechondrogenic cells in Bmprlb-null phalanges. This finding indicates that BMP signaling is necessary for phalangeal prechondrogenic cells to differentiate into chondrocytes and that signaling of BMP receptor 1B in this context is replaceable by that of a constitutively active BMP receptor 1A. These results suggest that BMP signaling in prechondrogenic cells and in growth plate chondrocytes stimulates their chondrocytic differentiation and maturation toward hypertrophy, respectively. bone morphogenetic protein receptor 1A | bone morphogenetic protein receptor 1B | bone morphogenetic protein | chondrocyte differentiation | transgenic

Stimulatory heterotrimeric G protein (Gs) transduces signals from various cell-surface receptors to adenylyl cyclases, which generate cAMP. The [alpha] subunit of Gs (Gs[alpha]) is encoded by GNAS (Gnas in mice), and heterozygous Gs[alpha] inactivating mutations lead to Albright hereditary osteodystrophy. The in vivo role of Gs[alpha] in skeletogenesis is largely unknown, because of early embryonic lethality of mice with disruption of Gnas exon 2 ([Gnas.sup.E2-/E2-]) and the absence of easily detectable phenotypes in growth plate chondrocytes of heterozygous mutant mice ([Gnas.sup.+/E2-]). We generated chimeric mice containing wild-type cells and either [Gnas.sup.E2-/E2-] or [Gnas.sup.+/ E2-]cells. [Gnas.sup.E2-/E2-] chondrocytes phenocopied PTH/PTHrP receptor [(PPR).sup.-/-] cells by prematurely undergoing hypertrophy. Introduction of a transgene expressing Gs[alpha] one of several gene products that include Gnas exon 2, into [Gnas.sup.E2-/E2-] cells prevented premature hypertrophy. Gs[alpha] mRNA expression detected by real-time RT-PCR analysis was reduced to approximately half that of the wild-type in both paternal and maternal [Gnas.sup.+/E2-] growth plate chondrocytes, indicating biallelic expression of Gs[alpha] in these cells. Hypertrophy of [Gnas.sup.+/E2-] chondrocytes was modestly but significantly premature in chimeric growth plates of mice containing wild-type and [Gnas.sup.+/E2-] cells. These data suggest that Gs[alpha] is the primary mediator of the actions of PPR in growth plate chondrocytes and that there is haploinsufficiency of Gs[alpha] signaling in [Gnas.sup.+/E2-] chondrocytes.

To determine the role of PTHrP in fetal calcium metabolism, blood calcium was measured in mice homozygous (HOM) for deletion of the PTHrP gene. On day 18.5 of gestation, ionized calcium and the maternal-fetal calcium gradient were significantly reduced in HOM PTHrP-ablated fetuses compared with that of their littermates. To assess the placental contribution to the effect of PTHrP, 45Ca and 51Cr-EDTA (as a blood diffusional marker) were administered by intracardiac injection to pregnant, heterozygous dams on day 17.5 of gestation. Five minutes after the injection, whole fetal 45Ca accumulation was significantly decreased in HOM PTHrP-ablated fetuses compared with that of their littermates. Next, two fetuses from each litter were injected in utero with fragments of PTHrP, PTH, or diluent 1 h before administering 45Ca and 51Cr to the dam. PTHrP-(1-86) and PTHrP-(67-86) significantly increased relative 45Ca accumulation in HOM PTHrP-ablated fetuses, but PTHrP-(1-34), PTH-(1-84), and the diluent had no effect. Finally, similar studies were performed on fetal mice that lacked the PTH/PTHrP receptor gene. Ionized calcium was significantly reduced in HOM PTH/PTHrP receptor-ablated fetuses. However, 5 min after maternal injection of 45Ca and 51Cr, relative accumulation of 45Ca was significantly increased in these fetuses. It was concluded that PTHrP is an important regulator of fetal blood calcium and placental calcium transport. In addition, the bioactivity of PTHrP for placental calcium transport is specified by a mid-molecular region that does not use the PTH/PTHrP receptor.

Nuclear extracts consisting of the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptor were investigated for binding to sequences in the 5'-flanking site of the human parathyroid hormone gene to identify DNA sequences that could mediate the transcriptional repression of a gene encoding parathyroid hormone. The sequences in the 25-base pair oligonucleotide, with sequences from -125 to -101 from the start of exon I, and binding nuclear proteins recognized by monoclonal bodies against the 1,25(OH)2D3 receptor, when positioned upstream to a heterologous viral promoter, mediate transcriptional repression in response to 1,25(OH)2D3 in GH4Cl cells, but not in ROS 17/2.8 cells.

Analysis of receptors for parathyroid hormone and parathyroid hormone related peptides expressed from cDNA obtained form rat osteosarcoma cells show these receptors are capable of stimulating multiple cellular enzymes. Results show that stimulation of these receptors lead to activation of adenylate cyclase and phospholipase C resulting in intracellular accumulation of cAMP and inositol triphosphate. This multiplicity of receptor function suggests that the various effects induced by parathyroid hormones can be mediated by a single receptor.

In the growth plate of endochondral bones, parathyroid hormone (PTH)-related peptide (PTHrP) regulates the rate of chondrocyte maturation from prehypertrophic chondrocytes to hypertrophic chondrocytes. Using an antibody specific for Sox9 phosphorylated at serine 181 ([S.sub.181]), one of the two consensus protein kinase A phosphorylation sites of Sox9, we showed that the addition of PTHrP strongly increased the phosphorylation of SOX9 in COS7 cells transfected with both SOX9- and PTH/PTHrP receptor-expressing vectors. PTHrP also increased the SOX9-dependent activity of chondrocyte-specific enhancers in the gene for type II collagen (Col2a1) in transient transfection experiments. This increased enhancer activity did not occur with a Sox9 mutant harboring serine-to-alanine substitutions in its two consensus protein kinase A phosphorylation sites. Consistent with these results, PTHrP also increased Col2a1 mRNA levels in rat chondrosarcoma cells as well as 10T1/2 mesenchymal cells transfected with a PTH/PTHrP receptor expressing plasmid. No phosphorylation of Sox9 at [S.sub.181] was detected in prehypertrophic chondrocytes of the growth plate or any chondrocytes of PTH/PTHrP receptor null mutants. In contrast in wild-type mouse embryos, previous immunohistochemistry experiments indicated that Sox9 phosphorylated at [S.sub.181] was detected almost exclusively in chondrocytes of the prehypertrophic zone. Sox9, regardless of the phosphorylation state, was present in all chondrocytes of both genotypes except hypertrophic chondrocytes. Our results indicated that Sox9 is a target of PTHrP signaling in prehypertrophic chondrocytes in the growth plate. We hypothesize that Sox9 mediates at least some effects of PTHrP in the growth plate and that the PTHrP-dependent increased transcriptional activity of Sox9 helps maintain the chondrocyte phenotype of cells in the prehypertrophic zone and inhibits their maturation to hypertrophic chondrocytes.

Formation of functional skeletal tissues requires highly organized steps of mesenchymal progenitor cell differentiation. The dental follicle (DF) surrounding the developing tooth harbors mesenchymal progenitor cells for various differentiated cells constituting the tooth root-bone interface and coordinates tooth eruption in a manner dependent on signaling by parathyroid hormone-related peptide (PTHrP) and the PTH/PTHrP receptor (PPR). However, the identity of mesenchymal progenitor cells in the DF and how they are regulated by PTHrP-PPR signaling remain unknown. Here, we show that the PTHrP-PPR autocrine signal maintains physiological cell fates of DF mesenchymal progenitor cells to establish the functional periodontal attachment apparatus and orchestrates tooth eruption. A single-cell RNA-seq analysis revealed cellular heterogeneity of PTHrP+ cells, wherein PTHrP+ DF subpopulations abundantly express PPR. Cell lineage analysis using tamoxifen-inducible PTHrP-creER mice revealed that PTHrP+ DF cells differentiate into cementoblasts on the acellular cementum, periodontal ligament cells, and alveolar cryptal bone osteoblasts during tooth root formation. PPR deficiency induced a cell fate shift of PTHrP+ DF mesenchymal progenitor cells to nonphysiological cementoblast-like cells precociously forming the cellular cementum on the root surface associated with up-regulation of Mef2c and matrix proteins, resulting in loss of the proper periodontal attachment apparatus and primary failure of tooth eruption, closely resembling human genetic conditions caused by PPR mutations. These findings reveal a unique mechanism whereby proper cell fates of mesenchymal progenitor cells are tightly maintained by an autocrine system mediated by PTHrP-PPR signaling to achieve functional formation of skeletal tissues. [ABSTRACT FROM AUTHOR]

Parathyroid hormone (PTH), the major calcium-regulating hormone, and norepinephrine (NE), the principal neurotransmitter of sympathetic nerves, regulate bone remodeling by activating distinct cell-surface G protein-coupled receptors in osteoblasts: the parathyroid hormone type 1 receptor (PTHR) and the β2-adrenergic receptor (β2AR), respectively. These receptors activate a common cAMP/PKA signal transduction pathway mediated through the stimulatory heterotrimeric G protein. Activation of β2AR via the sympathetic nervous system decreases bone formation and increases bone resorption. Conversely, daily injection of PTH (1-34), a regimen known as intermittent (i)PTH treatment, increases bone mass through the stimulation of trabecular and cortical bone formation and decreases fracture incidences in severe cases of osteoporosis. Here, we show that iPTH has no osteoanabolic activity in mice lacking the β2AR. β2AR deficiency suppressed both iPTH-induced increase in bone formation and resorption. We showed that the lack of β2AR blocks expression of iPTH-target genes involved in bone formation and resorption that are regulated by the cAMP/PKA pathway. These data implicate an unexpected functional interaction between PTHR and β2AR, two G protein-coupled receptors from distinct families, which control bone formation and PTH anabolism. [ABSTRACT FROM AUTHOR]

Parathyroid hormone (PTH)-related protein (PTHrP), regulated by Indian hedgehog and acting through the PTH/PTHrP receptor (PPR), is crucial for normal cartilage development. These observations suggest a possible role of PPR signaling in the postnatal growth plate; however, the role of PPR signaling in postnatal chondrocytes is unknown. In this study, we have generated tamoxifeninducible and cartilage-specific PPR KO mice to evaluate the physiological role of PPR signaling in postnatal chondrocytes. We found that inactivation of the PPR in chondrocytes postnatally leads to accelerated differentiation of chondrocytes, followed by disappearance of the growth plate. We also observed an increase of TUNEL-positive cells and activities of caspase-3 and caspase-9 in the growth plate, along with a decrease in phosphorylation of Bad at 5er155 in postnatal PPR KO mice. Administration of a low-phosphate diet, which prevents apoptosis of chondrocytes, prevented the disappearance of the growth plate. Taken together, these observations suggest that the major consequences of PPR activation are similar in both the fetal and postnatal growth plates. Moreover, chondrocyte apoptosis through the activation of a mitochondrial pathway may be involved in the process of premature disappearance of the growth plate by postnatal inactivation of the PPR in chondrocytes. [ABSTRACT FROM AUTHOR]

Formation of functional skeletal tissues requires highly organized steps of mesenchymal progenitor cell differentiation. The dental follicle (DF) surrounding the developing tooth harbors mesenchymal progenitor cells for various differentiated cells constituting the tooth root-bone interface and coordinates tooth eruption in a manner dependent on signaling by parathyroid hormone-related peptide (PTHrP) and the PTH/PTHrP receptor (PPR). However, the identity of mesenchymal progenitor cells in the DF and how they are regulated by PTHrP-PPR signaling remain unknown. Here, we show that the PTHrP-PPR autocrine signal maintains physiological cell fates of DF mesenchymal progenitor cells to establish the functional periodontal attachment apparatus and orchestrates tooth eruption. A single-cell RNA-seq analysis revealed cellular heterogeneity of PTHrP + cells, wherein PTHrP + DF subpopulations abundantly express PPR. Cell lineage analysis using tamoxifen-inducible PTHrP-creER mice revealed that PTHrP + DF cells differentiate into cementoblasts on the acellular cementum, periodontal ligament cells, and alveolar cryptal bone osteoblasts during tooth root formation. PPR deficiency induced a cell fate shift of PTHrP + DF mesenchymal progenitor cells to nonphysiological cementoblast-like cells precociously forming the cellular cementum on the root surface associated with up-regulation of Mef2c and matrix proteins, resulting in loss of the proper periodontal attachment apparatus and primary failure of tooth eruption, closely resembling human genetic conditions caused by PPR mutations. These findings reveal a unique mechanism whereby proper cell fates of mesenchymal progenitor cells are tightly maintained by an autocrine system mediated by PTHrP-PPR signaling to achieve functional formation of skeletal tissues., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Parathyroid hormone (PTH), the major calcium-regulating hormone, and norepinephrine (NE), the principal neurotransmitter of sympathetic nerves, regulate bone remodeling by activating distinct cell-surface G protein-coupled receptors in osteoblasts: the parathyroid hormone type 1 receptor (PTHR) and the β(2)-adrenergic receptor (β(2)AR), respectively. These receptors activate a common cAMP/PKA signal transduction pathway mediated through the stimulatory heterotrimeric G protein. Activation of β(2)AR via the sympathetic nervous system decreases bone formation and increases bone resorption. Conversely, daily injection of PTH (1-34), a regimen known as intermittent (i)PTH treatment, increases bone mass through the stimulation of trabecular and cortical bone formation and decreases fracture incidences in severe cases of osteoporosis. Here, we show that iPTH has no osteoanabolic activity in mice lacking the β(2)AR. β(2)AR deficiency suppressed both iPTH-induced increase in bone formation and resorption. We showed that the lack of β(2)AR blocks expression of iPTH-target genes involved in bone formation and resorption that are regulated by the cAMP/PKA pathway. These data implicate an unexpected functional interaction between PTHR and β(2)AR, two G protein-coupled receptors from distinct families, which control bone formation and PTH anabolism.