The procedures for bone and bone marrow section preparation, immunostaining conditions and antibodies are described in Supplementary Methods. The procedure for BrdU pulse labeling, LTR and subsequent detection has been reported 16 . The mice were fed BrdU (0.8 mg ml 21 in water) for 10 days, during which time 40% of LT-HSCs would divide at least once 31 . Seventy days after BrdU labelling, sections were stained with anti-BrdU antibody. N-cadherin 1 cell countFor quantitative analysis of N-cadherin þ cells, the sections were developed with AEC after being incubated with rabbit anti-N-cadherin antibody for 1 h and horseradish peroxidase (HRP)-conjugated goat anti-rabbit second antibody for 1 h. Three people counted the SNO cells in these sections, blind to the source of the sections. X-ray imageHigh-resolution X-rays (Faxitron MX-20) of bone and bone histomorphometry (OsteoMetrics, Inc.) were performed at the University of Missouri-Kansas City School of Dentistry. 1965-1972 (1996). 193-197 (2000). 10. Simmons, P., Gronthos, S. & Zannettino, A. C. Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals 1-3 . To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs) 4 . Here we show that PPRstimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, liganddependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by g-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.Mammalian bone marrow architecture involves haematopoietic stem cells (HSCs) in close proximity to the endosteal surfaces 5,6 , with more differentiated cells arranged in a loosely graduated fashion as the central longitudinal axis of the bone is approached 5,7,8 . This nonrandom organization of the marrow suggests a possible relationship between HSCs and osteoblasts-osteogenic cells lining the endosteal surface. Osteoblasts produce haematopoietic growth factors [9][10][11] and are activated by parathyroid hormone (PTH) or the locally produced PTH-related protein (PTHrP), through the PTH/ PTHrP receptor (PPR). We tested whether osteoblast...

PTH is a major systemic regulator of the concentrations of calcium, phosphate, and active vitamin D metabolites in blood and of cellular activity in bone. Intermittently administered PTH and amino-terminal PTH peptide fragments or analogs also augment bone mass and currently are being introduced into clinical practice as therapies for osteoporosis. The amino-terminal region of PTH is known to be both necessary and sufficient for full activity at PTH/PTHrP receptors (PTH1Rs), which mediate the classical biological actions of the hormone. It is well known that multiple carboxyl-terminal fragments of PTH are present in blood, where they comprise the major form(s) of circulating hormone, but these fragments have long been regarded as inert by-products of PTH metabolism because they neither bind to nor activate PTH1Rs. New in vitro and in vivo evidence, together with older observations extending over the past 20 yr, now points strongly to the existence of novel large carboxyl-terminal PTH fragments in blood and to receptors for these fragments that appear to mediate unique biological actions in bone. This review traces the development of this field in the context of the evolution of our understanding of the "classical" receptor for amino-terminal PTH and the now convincing evidence for these receptors for carboxyl-terminal PTH. The review summarizes current knowledge of the structure, secretion, and metabolism of PTH and its circulating fragments, details available information concerning the pharmacology and actions of carboxyl-terminal PTH receptors, and frames their likely biological and clinical significance. It seems likely that physiological parathyroid regulation of calcium and bone metabolism may involve receptors for circulating carboxy-terminal PTH ligands as well as the action of amino-terminal determinants within the PTH molecule on the classical PTH1R.

The linear sequence of intact mammalian PTH consists of 84 amino acids, of which only the most amino(N)-terminal portion, i.e. PTH-(1-34), is required for the classical actions of the hormone on mineral ion homeostasis mediated by the type 1 PTH/PTHrP receptor (PTH1R). Like the N-terminus, the carboxyl (C)-terminal sequence of PTH is highly conserved among species, and various circulating PTH C-fragments are generated by peripheral metabolism of intact PTH or are directly secreted, in a calcium-dependent manner, by the parathyroid glands. Certain synthetic PTH C-fragments exert actions on bone and cartilage cells that are not shared by PTH-(1-34), and specific binding of PTH C-peptides has been demonstrated in bone cells in which PTH1R expression was eliminated by gene targeting. The peptide human (h) PTH-(7-84) recently was shown to inhibit the calcemic actions of hPTH-(1-34) or hPTH-(1-84) in parathyroidectomized animals. To determine whether this anticalcemic effect of hPTH-(7-84) in vivo might result from direct actions on bone, we studied its effects on both resorption of intact bone in vitro and formation of osteoclasts in primary cultures of murine bone marrow. Human (h) PTH-(7-84) (300 nM) reduced basal 72-h release of preincorporated (45)Ca from neonatal mouse calvariae by 50% (9.6 +/- 1.9% vs. 17.8 +/- 5.7%; P < 0.001) and similarly inhibited resorption induced by hPTH-(1-84), hPTH-(1-34), 1,25-dihydroxyvitamin D(3) (VitD), PGE(2), or IL-11. In 12-d murine marrow cultures, both hPTH-(7-84) (300 nM) and hPTH-(39-84) (3000 nM) lowered VitD-dependent formation of osteoclast-like cells by 70%. On the contrary, these actions of hPTH-(7-84) were not observed with the PTH1R antagonists hPTH-(3-34)NH(2) and [L(11),D-W(12),W(23),Y(36)]hPTHrP-(7-36)NH(2), which, unlike hPTH-(7-84), did inhibit PTH1R-dependent cAMP accumulation in ROS 17/2.8 cells. We conclude that hPTH-(7-84), acting via receptors distinct from the PTH1R and presumably specific for PTH C-fragments, exerts a direct antiresorptive effect on bone that may be partly due to impaired osteoclast differentiation.

PTH is a potent systemic regulator of cellular differentiation and function in bone. It acts upon cells of the osteoblastic lineage via the G protein-coupled type-1 PTH/PTH-related peptide receptor (PTH1R). Carboxyl fragments of intact PTH(1-84) (C-PTH fragments) are cosecreted with it by the parathyroid glands in a calcium-dependent manner and also are generated via proteolysis of the hormone in peripheral tissues. Receptors that recognize C-PTH fragments (CPTHRs) have been described previously in osteoblastic and chondrocytic cells. To directly study CPTHRs in bone cells, we isolated clonal, conditionally transformed cell lines from fetal calvarial bone of mice that are homozygous for targeted ablation of the PTH1R gene and transgenically express a temperature-sensitive mutant SV40 T antigen. Cells with the highest specific binding of the CPTHR radioligand (125)I-[Tyr(34)]hPTH(19-84) exhibited a stellate, dendritic appearance suggestive of an osteocytic phenotype and expressed 6- to 10-fold more CPTHR sites/cell than did osteoblastic cells previously isolated from the same bones. In these osteocytic (OC) cells, expression of mRNAs for CD44, connexin 43, and osteocalcin was high, whereas that for alkaline phosphatase and cbfa-1/osf-2 was negligible. The CPTHR radioligand was displaced completely by hPTH(1-84), hPTH(19-84) and hPTH(24-84) (IC(50)s = 20-50 nM) and by hPTH(39-84) (IC(50) = 500 nM) but only minimally (24%) by 10,000 nM hPTH(1-34). CPTHR binding was down-regulated dose dependently by hPTH(1-84), an effect mimicked by ionomycin and active phorbol ester. Human PTH(1-84) and hPTH(39-84) altered connexin 43 expression and increased apoptosis in OC cells. Apoptosis induced by PTH(1-84) was blocked by the caspase inhibitor DEVD. We conclude that osteocytes, the most abundant cells in bone, may be principal target cells for unique actions of intact PTH(1-84) and circulating PTH C-fragments that are mediated by CPTHRs.

PTH regulates osteoblastic function by activating PTH/PTHrP receptors (PTH1Rs), which trigger several signaling pathways in parallel, including cAMP/protein kinase A (PKA) and, via both phospholipase-C (PLC)-dependent and PLC-independent mechanisms, protein kinase C (PKC). These signaling functions have been mapped to distinct domains within PTH(1-34), but their roles in mediating the anabolic effect of intermittent PTH in vivo are unclear. We compared the anabolic effects in mice of hPTH(1-34) with those of two analogs having restricted patterns of PTH1R signaling. [G 1 ,R 19 ]hPTH(1-28) lacks the 29-34 domain of hPTH(1-34) needed for PLCindependent PKC activation, incorporates a Gly 1 mutation that prevents PLC activation, and stimulates only cAMP/PKA signaling. [G 1 ,R 19 ]hPTH(1-34) retains the 29-34 domain and activates both cAMP/PKA and PLC-independent PKC.Human PTH(1-34) (40 μg/kg), [G 1 ,R 19 ]hPTH(1-34) (120 μg/kg), and [G 1 ,R 19 ]hPTH(1-28) (800 μg/kg), at doses equipotent in elevating blood cAMP at 10 min and cAMP-dependent gene expression in bone at 6 h after s.c. injection, were administered to 10 week old female C57BL/6J mice 5 days/ week for 4 weeks. Acute blood cAMP responses, retested after 4 weeks, were not reduced by the preceding PTH treatment. The three PTH peptides induced equivalent increases in distal femoral bone mineral density (BMD), and, by microCT analysis, distal femoral and vertebral bone volume and trabecular thickness and mid-femoral cortical endosteal apposition. [G 1 ,R 19 ]hPTH(1-34) and hPTH(1-34) increased distal femoral BMD more rapidly and augmented total-body BMD and bone volume of proximal tibial trabeculi to a greater extent than did [G 1 ,R 19 ]hPTH(1-28),.We conclude that cAMP/PKA signaling is the dominant mechanism for the anabolic actions of PTH in trabecular bone and PLC-independent PKC signaling, attributable to the PTH(29-34) sequence, appears to accelerate the trabecular response and augment BMD at some skeletal sites. PTH1R PLC signaling pathway is not required for an anabolic effect of intermittent PTH(1-34) on bone.

The bone morphogenetic proteins (BMPs) play a pivotal role in endochondral bone formation. Using differential display polymerase chain reaction, we have identified a novel gene, named BIG-3 (BMP-2-induced gene 3 kb), that is induced as a murine prechondroblastic cell line, MLB13MYC clone 17, acquires osteoblastic features in response to BMP-2 treatment. The 3-kilobase mRNA encodes a 34-kDa protein containing seven WD-40 repeats. Northern and Western analyses demonstrated that BIG-3 mRNA and protein were induced after 24 h of BMP-2 treatment. BIG-3 mRNA was expressed in conditionally immortalized murine bone marrow stromal cells, osteoblasts, osteocytes, and growth plate chondrocytes, as well as in primary calvarial osteoblasts. Immunohistochemistry demonstrated that BIG-3 was expressed in the osteoblasts of calvariae isolated from mouse embryos. To identify a role for BIG-3 in osteoblast differentiation, MC3T3-E1 cells were stably transfected with the full-length coding region of BIG-3 (MC3T3E1-BIG-3) cloned downstream of a cytomegalovirus promoter in pcDNA3.1. Pooled MC3T3E1-BIG-3 clones expressed alkaline phosphatase activity earlier and achieved a peak level of activity 10-fold higher than cells transfected with the empty vector (MC3T3E1-EV) at 14 days. Cyclic AMP production in response to parathyroid hormone was increased 10-and 14-fold at 7 and 14 days, respectively, in MC3T3E1-BIG-3 clones, relative to MC3T3E1-EV clones. This increase in cAMP production was associated with an increase in PTH binding. Expression of BIG-3 increased mRNA levels encoding Cbfa1, type I collagen, and osteocalcin and accelerated formation of mineralized nodules. In conclusion, we have identified a novel WD-40 protein, induced by BMP-2 treatment, that dramatically accelerates the program of osteoblastic differentiation in stably transfected MC3T3E1 cells.

Previous studies have shown that mice missing gp130, the common receptor subunit for many cytokines, die at or before birth with multiple skeletal abnormalities. Furthermore, interactions between PTH and gp130 signaling have suggested that gp130 signaling might influence calcium homeostasis. We, therefore, examined the function of osteoblasts, osteoclasts, and calcium homeostasis in gp130(-/-) mice, both in vivo and in vitro. Osteoblasts from these mice exhibit widespread abnormalities, including decreased alkaline phosphatase mRNA and protein, both in vivo and in osteoblast cultures. Although osteoclast number is increased in gp130(-/-) fetuses, these osteoclasts exhibit abnormalities in the resorptive organelle and the ruffled border, and the mice are mildly hypocalcemic. Although the hypocalcemia is associated with secondary hyperparathyroidism, the increase in PTH does not explain the increase in osteoclast number because removal of the PTH gene in gp130(-/-) fetuses does not importantly change osteoclast number. Calvarial bone resorption in response to PTH is defective, as is the ability of osteoblastic cells from gp130(-/-) mice to stimulate osteoclastogenesis from normal precursors in vitro or to increase receptor activator of nuclear factor-kappa B ligand mRNA levels after exposure to PTH. These studies demonstrate the importance of gp130 signaling for osteoblast function and calcium homeostasis.

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