Jude E. Onyia

Ghrelin octanoylation mediated by an orphan lipid transferase

The peptide hormone ghrelin is the only known protein modified with an O-linked octanoyl side group, which occurs on its third serine residue. This modification is crucial for ghrelins physiological effects including regulation of feeding, adiposity, and insulin secretion. Despite the crucial role for octanoylation in the physiology of ghrelin, the lipid transferase that mediates this novel modification has remained unknown. Here we report the identification and characterization of human GOAT, the ghrelin O-acyl transferase. GOAT is a conserved orphan membrane-bound O-acyl transferase (MBOAT) that specifically octanoylates serine-3 of the ghrelin peptide. Transcripts for both GOAT and ghrelin occur predominantly in stomach and pancreas. GOAT is conserved across vertebrates, and genetic disruption of the GOAT gene in mice leads to complete absence of acylated ghrelin in circulation. The occurrence of ghrelin and GOAT in stomach and pancreas tissues demonstrates the relevance of GOAT in the acylation of ghrelin and further implicates acylated ghrelin in pancreatic function.

Catabolic Effects of Continuous Human PTH (1–38) in Vivo Is Associated with Sustained Stimulation of RANKL and Inhibition of Osteoprotegerin and Gene-Associated Bone Formation

Continuous infusion of PTH in vivo results in active bone resorption. To investigate the molecular basis of the catabolic effect of PTH in vivo, we evaluated the role of OPG and RANKL, which are known to influence osteoclast formation and function. Weanling rats fed a calcium-free diet were parathyroidectomized and infused with PTH via an Alzet pump to examine: 1) the changes of serum-ionized calcium and osteoclast number, 2) the expression of OPG/RANKL mRNA and protein, and 3) the expression of osteoblast phenotype bone formation-associated genes such as osteoblast specific transcription factor, osteocalcin, bone sialoprotein, and type I collagen. PTH (1–38) (0.01–20 μg/100 g) continuous infusion for 1–24 h resulted in a dose-dependent increase in serum-ionized calcium in parathyroidectomized rats and a corresponding dose-dependent increase in osteoclast number, indicating an increased bone resorption. At 20 μg/100 g PTH dose level, serum-ionized calcium was 2.1-fold of the vehicle control and not differ...

Osteoblast Apoptosis and Bone Turnover

With the discoveries of different death mechanisms, an emerging definition of apoptosis is the process of cell death associated with caspase activation or caspase‐mediated cell death. This definition accepts that caspases represent the final common mechanistic pathway in apoptosis. Apoptosis may be triggered either by activation events that target mitochondria or endoplasmic reticulum or by activation of cell surface “death receptors,” for example, those in the tumor necrosis factor (TNF) superfamily. In the postnatal and adult skeleton, apoptosis is integral to physiological bone turnover, repair, and regeneration. The balance of osteoblast proliferation, differentiation, and apoptosis determines the size of the osteoblast population at any given time. Although apoptosis has been recorded in many studies of bone, the selective mechanisms invoked in the different models studied rarely have been identified. This review offers a broad overview of the current general concepts and controversies in apoptosis research and then considers specific examples of osteoblast apoptosis pertinent to skeletal development and to the regulation of bone turnover. In reviewing selected work on interdigital apoptosis in the developing skeleton, we discuss the putative roles of the bone morphogenetic proteins (BMPs), Msx2, RAR‐γ, and death inducer obliterator 1 (DIO‐1). In reviewing factors regulating apoptosis in the postnatal skeleton, we discuss roles of cytokines, growth factors, members of the TNF pathway, and the extracellular matrix (ECM). Finally, the paradoxical effects of parathyroid hormone (PTH) on osteoblast apoptosis in vivo are considered in the perspective of a recent hypothesis speculating that this may be a key mechanism to explain the anabolic effects of the hormone. An improved understanding of the apoptotic pathways and their functional outcomes in bone turnover and fracture healing may facilitate development of more targeted therapeutics to control bone balance in patients with osteoporosis and other skeletal diseases.

Effects of parathyroid hormone on Wnt signaling pathway in bone

The Wnt signaling pathway has recently been demonstrated to play an important role in bone cell function. In previous studies using DNA microarray analyses, we observed a change in some of the molecular components of the canonical Wnt pathway namely, frizzled‐1 (FZD‐1) and axil, in response to continuous parathyroid hormone (PTH) treatment in rats. In the present study, we further explored other components of the Wnt signaling pathway in rat distal metaphyseal bone in vivo, and rat osteoblastic osteosarcoma cells (UMR 106) in culture. Several Wnt pathway components, including low‐density lipoprotein‐receptor‐related protein 5 (LRP5), LRP6, FZD‐1, Dickkopf‐1 (Dkk‐1), and Kremen‐1 (KRM‐1), were expressed in bone in vivo and in osteoblasts in vitro. Continuous exposure to PTH (1–38) both in vivo and in vitro upregulated the mRNA expression of LRP6 and FZD‐1 and decreased LRP5 and Dkk‐1. These effects in UMR 106 cells were associated with an increase in β‐catenin as measured by Western blots and resulted in functional activation (three to six‐fold) of a downstream Wnt responsive TBE6‐luciferase (TCF/LEF‐binding element) reporter gene. Activation of the TBE6‐luciferase reporter gene by PTH (1–38) in UMR 106 cells was inhibited by the protein kinase A (PKA) inhibitor, H89. Activation was mimicked by PTH (1–31), PTH‐related protein (1–34), and forskolin, but both PTH (3–34) and (7–34) had no effect. These findings suggest that the effect of PTH on the canonical Wnt signaling pathway occurs at least in part via the cAMP‐PKA pathway through the differential regulation of the receptor complex proteins (FZD‐1/LRP5 or LRP6) and the antagonist (Dkk‐1). Taken together, these results reveal a possible role for the Wnt signaling pathway in PTH actions in bone.

The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function.

Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblast-secreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE2)), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the β-galactosidase reporter gene (pOPG5.9βgal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9βgal reporter construct led to 39-, 7-, and 16-fold increases in β-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE2 elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE2 element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequence-specific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.

Orally bioavailable GSK-3α/β dual inhibitor increases markers of cellular differentiation in vitro and bone mass in vivo

GSK‐3, a component of the canonical Wnt signaling pathway, is implicated in regulation of bone mass. The effect of a small molecule GSK‐3 inhibitor was evaluated in pre‐osteoblasts and in osteopenic rats. GSK‐3 inhibitor induced osteoblast differentiation in vitro and increased markers of bone formation in vitro and in vivo with concomitant increased bone mass and strength in rats.

In vivo, human parathyroid hormone fragment (hPTH 1–34) transiently stimulates immediate early response gene expression, but not proliferation, in trabecular bone cells of young rats

Intermittent PTH increases trabecular bone mass in vivo by stimulating osteoblast differentiation to increase bone formation. The molecular events that mediate the anabolic effect of PTH on osteoblasts have not been characterized. We investigated if PTH regulated mRNA expression of proto-oncogenes, c-fos, c-jun, and c-myc, early response genes that have been shown to be involved in the regulation of both cell proliferation and differentiation. As PTH also regulates the early expression of the cytokine, interleukin-6 (IL-6), in bone cells in vitro, we also investigated if this occurred in vivo, in concert with the other early response genes. Northern blot hybridization was used to analyze mRNA expression in the metaphysis of the distal femur of young rats. To determine the proliferative state in these femurs, mRNA expression of the cell proliferation marker histone, H4, was assessed. Subcutaneous administration of a single injection of human PTH (1-34) at 8 micrograms/100 g, a dose known to increase bone forming surfaces, induced rapid and transient expression of c-fos, c-jun, c-myc, and IL-6 mRNA. A second novel transcript for IL-6 was detected, but its significance remains unknown. Induction of all these messages was evident by 1 h; the levels of mRNA returned to baseline after 3-6 h. Concurrently, PTH had a small inhibitory effect on the expression of histone H4 mRNA. We conclude that, in vivo, PTH upregulates cell differentiation in trabecular bone by transient stimulation of the early response genes and IL-6, while downregulating cell proliferation.

EphrinB2 regulation by PTH and PTHrP revealed by molecular profiling in differentiating osteoblasts.

With the aim of identifying new pathways and genes regulated by PTH(1–34) and PTH‐related protein 1–141 [PTHrP(1–141)] in osteoblasts, this study was carried out using a mouse marrow stromal cell line, Kusa 4b10, that acquires features of the osteoblastic phenotype in long‐term culture conditions. After the appearance of functional PTH receptor 1 (PTHR1) in Kusa 4b10 cells, they were treated with either PTH(1–34) or PTHrP(1–141), and RNA was subjected to Affymetrix whole mouse genome array. The microarray data were validated using quantitative real‐time RT‐PCR on independently prepared RNA samples from differentiated Kusa 4b10, UMR106 osteosarcoma cells, and primary mouse calvarial osteoblasts, as well as in vivo using RNA from metaphyseal bone after a single PTH injection to 3‐wk‐old and 6‐mo‐old ovariectomized rats. Of the 45,101 probes used on the microarray, 4675 were differentially expressed by ≥1.5 fold, with a false discovery rate <0.1. Among the regulated genes, ephrinB2 mRNA was upregulated in response to both PTH and PTHrP. This was confirmed by quantitative real‐time PCR in vitro and in vivo. Increased ephrinB2 protein was also shown in vitro by Western blotting, and immunostaining of femur sections showed ephrinB2 in both osteoclasts and osteoblasts. Production of ephrinB2, as well as other ephrins or Eph family members, did not change during differentiation of Kusa 4b10 cells. Blockade of ephrinB2/EphB4 interaction resulted in inhibition of mineralization of Kusa 4b10 cells. Together with the shown effect of ephrinB2 promoting osteoblast differentiation and bone formation through action on EphB4, the data raise the possibility that PTH or PTHrP might regulate ephrinB2 to act in a paracrine or autocrine manner on EphB4 or EphB2 in the osteoblast, contributing as a local event to the anabolic action of PTH or PTHrP.

Activation of osteocalcin transcription involves interaction of protein kinase A- and protein kinase C-dependent pathways.

Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, synthesized and secreted exclusively by osteoblastic cells in the late stage of maturation, and is considered indicator of osteoblast differentiation. Osteocalcin expression is modulated by parathyroid hormone (PTH) and a variety of other factors. The cAMP-dependent protein kinase pathway has been shown previously to have an essential role in PTH signaling and regulation of osteocalcin expression. To determine the extent to which other pathways may also participate in osteocalcin expression, we used rat and human osteoblast-like cell lines to generate stably transfected clones in which the osteocalcin promoter was fused to a luciferase reporter gene. These clones were examined for their responsiveness to agents known to activate or interfere with protein kinase A (PKA)- and protein kinase C (PKC)-dependent pathways. We have found that forskolin, cAMP, and PTH, as well as insulin-like growth factor I (IGF-I) and basic fibroblast growth factor, all were effective in activating the osteocalcin promoter. Phorbol 12-myristate 13-acetate (PMA) was also a strong inducer of the promoter, indicating that PKC plays a role in expression of osteocalcin. In combination with PTH or forskolin, the effect of PMA was additive to synergistic. Calphostin C, a selective inhibitor of PKC, decreased the PMA-, PTH-, and IGF-I-induced luciferase activity in a dose-dependent manner; a PKA inhibitor, H-89, also blocked the induction by PTH and IGF-I but not by PMA. We conclude that regulation of osteocalcin transcription is mediated by both PKA-dependent and PKC-dependent mechanisms and that the respective kinases reside on a linear or convergent pathway.

Calcitonin impairs the anabolic effect of PTH in young rats and stimulates expression of sclerostin by osteocytes

The therapeutic goal of increasing bone mass by co-treatment of parathyroid hormone (PTH) and an osteoclast inhibitor has been complicated by the undefined contribution of osteoclasts to the anabolic activity of PTH. To determine whether active osteoclasts are required at the time of PTH administration, we administered a low dose of the transient osteoclast inhibitor salmon calcitonin (sCT) to young rats receiving an anabolic PTH regimen. Co-administration of sCT significantly blunted the anabolic effect of PTH as measured by peripheral quantitative computer tomography (pQCT) and histomorphometry in the femur and tibia, respectively. To determine gene targets of sCT, we carried out quantitative real time PCR and microarray analysis of metaphyseal samples 1.5, 4 and 6.5h after administration of a single injection of PTH, sCT or PTH+sCT. Known targets of PTH action, IL-6, ephrinB2 and RANKL, were not modified by co-administration with sCT. Surprisingly, at all time points, we noted a significant upregulation of sclerostin mRNA by sCT treatment, as well as down-regulation of two other osteocyte gene products, MEPE and DMP1. Immunohistochemistry confirmed that sCT administration increased the percentage of osteocytes expressing sclerostin, suggesting a mechanism by which sCT reduced the anabolic effect of PTH. Neither mRNA for CT receptor (Calcr) nor labeled CT binding could be detected in sclerostin-enriched cells differentiated from primary calvarial osteoblasts. In contrast, osteocytes freshly isolated from calvariae expressed a high level of Calcr mRNA. Furthermore immunohistochemistry revealed co-localization of CT receptor (CTR) and sclerostin in some osteocytes in calvarial sections. Taken together these data indicate that co-treatment with sCT can blunt the anabolic effect of PTH and this may involve direct stimulation of sclerostin production by osteocytes. These data directly implicate calcitonin as a negative regulator of bone formation through a previously unsuspected mechanism.