F. Richard Bringhurst

Regulation of C-terminal and intact FGF-23 by dietary phosphate in men and women.

FGF‐23 is a novel regulator of phosphate metabolism. We studied the regulation of FGF‐23 by dietary phosphate in 66 men and women using two assays. Dietary phosphate restriction decreased FGF‐23 and loading increased FGF‐23 significantly. An assay that measured intact FGF‐23 showed the effects of dietary phosphate much more clearly than an assay that also measures presumed biologically inactive fragments. Dietary phosphate is a key regulator of circulating FGF‐23; choice of assay is critical when studying FGF‐23 physiology.

Targeted ablation of the PTH/PTHrP receptor in osteocytes impairs bone structure and homeostatic calcemic responses

Parathyroid hormone (PTH) is a major physiologic regulator of calcium, phosphorous, and skeletal homeostasis. Cells of the osteoblastic lineage are key targets of PTH action in bone, and recent evidence suggests that osteocytes might be important in the anabolic effects of PTH. To understand the role of PTH signaling through the PTH/PTHrP receptors (PPR) in osteocytes and to determine the role(s) of these cells in mediating the effects of the hormone, we have generated mice in which PPR expression is specifically ablated in osteocytes. Transgenic mice in which the 10 kb-Dmp1 promoter drives a tamoxifen-inducible Cre-recombinase were mated with animals in which exon 1 of PPR is flanked by lox-P sites. In these animals, osteocyte-selective PPR knockout (Ocy-PPR(cKO) mice) could be induced by administration of tamoxifen. Histological analysis revealed a reduction in trabecular bone and mild osteopenia in Ocy-PPR(cKO) mice. Reduction of trabeculae number and thickness was also detected by micro-computed tomography analysis whereas bone volume fraction (BV/TV%) was unchanged. These findings were associated with an increase in Sost and sclerostin expression. When Ocy-PPR(cKO) mice were subjected to a low-calcium diet to induce secondary hyperparathyroidism, their blood calcium levels were significantly lower than littermate controls. Moreover, PTH was unable to suppress Sost and sclerostin expression in the Ocy-PPR(cKO) animals, suggesting an important role of PTH signaling in osteocytes for proper bone remodeling and calcium homeostasis.

Cyclic adenosine monophosphate/protein kinase A mediates parathyroid hormone/parathyroid hormone-related protein receptor regulation of osteoclastogenesis and expression of RANKL and osteoprotegerin mRNAs by marrow stromal cells.

Parathyroid hormone (PTH) is a major regulator of osteoclast formation and activation, effects that are associated with reciprocal up‐ and down‐regulation of RANKL and osteoprotegerin (OPG), respectively. The roles of specific downstream signals generated by the activated PTH/PTH‐related protein (PTHrP) receptor (PTH1R), such as cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and phospholipase C/protein kinase C (PLC/PKC), in controlling RANKL and OPG expression and osteoclastogenesis remain uncertain. In MS1 conditionally transformed clonal murine marrow stromal cells, which support PTH‐induced osteoclast formation from cocultured normal spleen cells, PTH(1–34) increased RANKL and macrophage colony‐stimulating factor (M‐CSF) mRNA expression and decreased that of OPG when present continuously for 7–20 days at 37°C in the presence of dexamethasone (Dex). In cells precultured for 7 days and then treated with PTH(1–34), similar reciprocal regulation of RANKL and OPG occurred, maximally at 6–24 h, that was of greater amplitude than the changes induced by chronic (7–10 days) PTH exposure. These acute effects of PTH(1–34) were mimicked by PKA stimulators (8‐bromoadenosine [8Br]‐cAMP or forskolin [FSK]), blocked by the PKA inhibitor Rp‐cAMPs but unaffected by the PKC inhibitor GF109203X. Amino‐truncated PTH(1–34) analogs PTH(5–34) and PTH(7–34) neither increased cAMP production in MS1 cells nor regulated RANKL or OPG mRNA. Reciprocal RANKL/OPG mRNA regulation was induced in MS1 cells by PTH(3–34) but only at high concentrations that also increased cAMP. The highly PKA‐selective PTH analog [Gly1,Arg19]human PTH(1–28) exerted effects similar to PTH(1–34) on RANKL and OPG mRNAs and on osteoclast formation, both in MS1/spleen cell cocultures and in normal murine bone marrow cultures. The direct PKC stimulator 12‐O‐tetradecanoylphorbol‐13‐acetate (PMA) did not induce RANKL mRNA in MS1 cells, but it did up‐regulate OPG mRNA and also antagonized osteoclast formation induced by PTH(1–34) in both MS1/spleen cocultures and normal bone marrow cultures. Thus, cAMP/PKA signaling via the PTH1R is the primary mechanism for controlling RANKL‐dependent osteoclastogenesis, although direct PKC activation may negatively regulate this effect of PTH by inducing expression of OPG.

Mutations in the Second Cytoplasmic Loop of the Rat Parathyroid Hormone (PTH)/PTH-related Protein Receptor Result in Selective Loss of PTH-stimulated Phospholipase C Activity

To define the structural requirements of the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor necessary for activation of phospholipase C (PLC), receptors with random mutations in their second cytoplasmic loop were synthesized, and their properties were assessed. A mutant in which the wild type (WT) rat PTH/PTHrP receptor sequence EKKY (amino acids 317-320) was replaced with DSEL had little or no PTH-stimulated PLC activity when expressed transiently in COS-7 cells, but it retained full capacity to bind ligand and to generate cAMP. This phenotype was confirmed in LLC-PK1 cells stably expressing the DSEL mutant receptor, where both PTH-stimulated PLC activity and sodium-dependent phosphate co-transport were essentially abolished. Individual mutations of these four residues point to a critical role for Lys-319 in receptor-G protein coupling. PTH-generated IPs were reduced to 27 ± 13% when K319E, compared with the WT receptor, and PLC activation was fully recovered in a receptor revertant in which Glu-319 in the DSEL mutant cassette was restored to the WT residue, Lys. Moreover, the WT receptor and a mutant receptor in which K319R had indistinguishable properties, thus suggesting that a basic amino acid at this position may be important for PLC activation. All of these receptors had unimpaired capacity to bind ligand and to generate cAMP. To ensure adequacy of Gαq-subunits for transducing the receptor signal, Gαq was expressed in HEK293 and in LLC-PK1 cells together with either WT receptors or receptors with the DSEL mutant cassette. PTH generated no inositol phosphates (IPs) in either HEK293 or LLC-PK1 cells, when they expressed DSEL mutant receptors together with Gαq. In contrast, PTH generated 2- and 2.5-fold increases in IPs, respectively, when these cells co-expressed both the WT receptor and Gαq. Thus, generation of IPs by the activated PTH/PTHrP receptor can be selectively abolished without affecting its capacity to generate cAMP, and Lys-319 in the second intracellular loop is critical for activating the PLC pathway. Moreover, α-subunits of the Gq family, rather than βγ-subunits, transduce the signal from the activated receptor to PLC, and the PLC, rather than the adenylyl cyclase, pathway mediates sodium-dependent phosphate co-transport in LLC-PK1 cells.

Loss of wnt/β-catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes.

Wnt signaling is essential for osteogenesis and also functions as an adipogenic switch, but it is not known if interrupting wnt signaling via knockout of β‐catenin from osteoblasts would cause bone marrow adiposity. Here, we determined whether postnatal deletion of β‐catenin in preosteoblasts, through conditional cre expression driven by the osterix promoter, causes bone marrow adiposity. Postnatal disruption of β‐catenin in the preosteoblasts led to extensive bone marrow adiposity and low bone mass in adult mice. In cultured bone marrow–derived cells isolated from the knockout mice, adipogenic differentiation was dramatically increased, whereas osteogenic differentiation was significantly decreased. As myoblasts, in the absence of wnt/β‐catenin signaling, can be reprogrammed into the adipocyte lineage, we sought to determine whether the increased adipogenesis we observed partly resulted from a cell‐fate shift of preosteoblasts that had to express osterix (lineage‐committed early osteoblasts), from the osteoblastic to the adipocyte lineage. Using lineage tracing both in vivo and in vitro we showed that the loss of β‐catenin from preosteoblasts caused a cell‐fate shift of these cells from osteoblasts to adipocytes, a shift that may at least partly contribute to the bone marrow adiposity and low bone mass in the knockout mice. These novel findings indicate that wnt/β‐catenin signaling exerts control over the fate of lineage‐committed early osteoblasts, with respect to their differentiation into osteoblastic versus adipocytic populations in bone, and thus offers potential insight into the origin of bone marrow adiposity.

The PTH/PTHrP Receptor Can Delay Chondrocyte Hypertrophy In Vivo without Activating Phospholipase C

One G protein-coupled receptor (GPCR) can activate more than one G protein, but the physiologic importance of such activation has not been demonstrated in vivo. We have generated mice expressing exclusively a mutant form of the PTH/PTHrP receptor (DSEL) that activates adenylyl cyclase normally but not phospholipase C (PLC). DSEL mutant mice exhibit abnormalities in embryonic endochondral bone development, including delayed ossification and increased chondrocyte proliferation. Analysis of the differentiation of embryonic metatarsals in vitro shows that PTH(1-34) and forskolin inhibit, whereas active phorbol ester stimulates, hypertrophic differentiation. Thus, PLC signaling via the PTH/PTHrP receptor normally slows the proliferation and hastens the differentiation of chondrocytes, actions that oppose the dominant effects of PTH/PTHrP receptors and that involve cAMP-dependent signaling pathways.

Dual Signaling and Ligand Selectivity of the Human PTH/PTHrP Receptor

Parathyroid hormone (PTH) activates PTH/PTH‐related peptide‐related receptors (PTHRs) to stimulate both adenylyl cyclase (AC) and phospholipase C (PLC). How these parallel signals mediate specific cellular and tissue responses to PTH, such as the complex anabolic versus catabolic actions of PTH on bone, remains unsettled. Previous studies of PTHR signaling and function employed mainly rodent or other cell lines that express endogenous PTHRs and, possibly, alternate species of PTH receptors. To preclude confounding effects of such receptors, we stably expressed recombinant human PTHRs (hPTHRs) at different levels of surface density in LLC‐PK1 porcine renal epithelial cells that lack endogenous PTH responsiveness. hPTH(1–34) induced concentration‐dependent activation of both AC and PLC via transfected hPTHRs. Maximal intensity of each signal increased with receptor density, but more hPTHRs were required for PLC than for AC activation. Coupling to AC was saturated at receptor densities too low to detect sustained PLC activation. hPTH(3–34), found by others to be a PLC/protein kinase C (PKC)‐selective peptide in rat cells, did not activate PLC via human (or rat) PTHRs under conditions (1 μM peptide, 106 hPTHRs/cell) where hPTH(1–34) stimulated PLC severalfold. Other cellular responses that require PKC activation in these cells, such as sodium‐dependent phosphate transport and cAMP‐independent secretion of plasminogen activator, were induced by PTH(1–34) but not by hPTH(3–34) or hPTH(7–34). We conclude that amino‐truncated PTH analogs reported to activate PKC cannot directly activate phosphatidylinositol‐specific PLC via the human or rat PTHR and therefore that PTH receptors may access alternate, PLC‐independent pathways of PKC activation in some target cells. The relative intensity of AC and PLC signaling via the hPTHR may be strongly regulated by changes in its surface expression.

Effects of vitamin D metabolites and analogs on bone collagen synthesis in vitro

SummaryThe effects of selected vitamin D3 metabolites and analogs on bone collagen synthesis in vitro were examined in organ cultures of neonatal mouse calvarial bone. The incorporation of [3H]proline into the collagenase-digestible fraction of newly synthesized protein was progressively inhibited by 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) (10−12 M to 10−7 M) in 24-h cultures, and incorporation into noncollagen protein was also blunted at the higher doses employed. The synthetic analog 1α-hydroxyvitamin D3 (1α-OHD3) was almost 300-fold less potent an inhibitor of collagen synthesis than was 1α,25(OH)2D3, and the natural metabolites 25-hydroxyvitamin D3 (25OHD3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1000-fold less potent, although the dose-response curve for each of these compounds was not parallel with that for 1α,25(OH)2D3. The 24S,25(OH)2D3 enantiomer was four-fold less potent than 24R,25-(OH)2D3 or 25OHD3, and vitamin D3 showed less than 2% the activity of 25OHD3. The responses were unaffected by the substitution of 0.4% bovine albumin for 5% horse serum in the medium, and no stimulation of collagen synthesis was observed in response to 25-hydroxylated metabolites between 2×10−14 and 2×10−6 M or in cultures treated for up to 96 h with 24R,25(OH)2D3 (2×10−10M).The overall results emphasize the similarity of the structural requirements for the inhibition of matrix synthesis and the stimulation of resorption by active vitamin D metabolites in bone. In addition, these studies support the importance of the 1-hydroxyl function to the biologic activity of vitamin D in the skeleton.

Phospholipase C Signaling via the Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Is Essential for Normal Bone Responses to PTH

We have previously shown that differentiation of hypertrophic chondrocytes is delayed in mice expressing a mutated PTH/PTHrP receptor (PTHR) (called DSEL here) that stimulates adenylyl cyclase normally but fails to activate phospholipase C (PLC). To better understand the role of PLC signaling via the PTHR in skeletal and mineral homeostasis, we examined these mice fed a normal or calcium-deficient diet. On a standard diet, DSEL mice displayed a modest decrease in bone mass. Remarkably, when fed a low-calcium diet or infused with PTH, DSEL mice exhibited strikingly curtailed peritrabecular stromal cell responses and attenuated new bone formation when compared with Wt mice. Attenuated in vitro colony formation was also observed in bone marrow cells derived from DSEL mice fed a low-calcium diet. Furthermore, PTH stimulated proliferation and increased mRNAs encoding cyclin D1 in primary osteoblasts derived from Wt but not from DSEL mice. Our data indicate that PLC signaling through the PTHR is required for skeletal homeostasis.

Type-1 parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptors activate phospholipase C in response to carboxyl-truncated analogs of PTH(1-34).

The carboxyl(C)-truncated human (h) PTH (hPTH) analog hPTH(1–31), which activates adenylyl cyclase (AC), but not protein kinase C, in rat osteosarcoma cells, exerts an anabolic effect on rat bone in vivo similar to that of hPTH(1–34). It has been proposed, therefore, that this action of PTH(1–34) is mediated exclusively by stimulation of AC via the rat type-1 PTH/PTH-related peptide (PTHrP) receptor (PTH1R). To determine whether this selective signaling pattern also might be a property of the hPTH1R, we studied signal transduction via heterologously expressed hPTH1Rs in response to activation by hPTH(1–34), hPTH(1–31), and a C-truncated analog that does not increase rat bone mass in vivo, hPTH(1–30). In porcine LLC-PK1 cells that stably expressed recombinant hPTH1Rs, these three peptides activated AC identically (EC50 = 1–2 nm). In cells with comparable expression of rat PTH1Rs, AC activation by hPTH(1–34) and hPTH(1–31) again was identical, whereas full activation by hPTH(1–30) required higher concentrat...