The Education Review Board: A Mechanism for Managing Potential Conflicts of Interest in Medical Education.

Concerns about the influence of industry support on medical education, research, and patient care have increased in both medical and political circles. Some academic medical centers, questioning whether industry support of medical education could be appropriate and not a conflict of interest, banned such support. In 2009, a Partners HealthCare System commission concluded that interactions with industry remained important to Partners' charitable academic mission and made recommendations to transparently manage such relationships. An Education Review Board (ERB) was created to oversee and manage all industry support of Partners educational activities.Using a case review method, the ERB developed guidelines to implement the commission's recommendations. A multi-funder rule was established that prohibits industry support from only one company for any Partners educational activity. Within that framework, the ERB established guidelines on industry support of educational conferences, clinical fellowships, and trainees' expenses for attending external educational programs; gifts of textbooks and other educational materials; promotional opportunities associated with Partners educational activities; Partners educational activities under contract with an industry entity; and industry-run programs using Partners resources.Although many changes have resulted from the implementation of the ERB guidelines, the number of industry grants for Partners educational activities has remained relatively stable, and funding for these activities declined only moderately during the first three full calendar years (2011-2013) of ERB oversight. The ERB continually educates both the Partners community and industry about the rationale for its guidelines and its openness to their refinement in response to changes in the external environment.

Nuclear translocation of CBP/p300-interacting protein CITED1 induced by parathyroid hormone requires serine phosphorylation at position 79 in its 63-84 domain.

The transcriptional cofactor CITED1 inhibits osteoblastic differentiation and blunts the stimulation of osteoblastic differentiation by parathyroid hormone (PTH). In the MC3T3-E1 osteoblastic cell line, we found that CITED1 was located predominantly in the cytoplasm and that hPTH(1-34) increased translocation of CITED1 from the cytoplasm to the nucleus. This response to hPTH(1-34) was not observed when all 9 serine residues within the 63-84 domain of CITED1 were mutated to alanines (CITED1 9S>A) or when a single serine to alanine mutation was made at position 79 (CITED1 S(79)>A). CITED1 containing mutations of these 9 serines to glutamic acid (9S>E) retained the same nuclear translocation response to hPTH(1-34) as the wild type CITED1. ALP activity and formation of mineralized nodules were inhibited in cells transfected with pcDNA3-CFP-CITED1 or with pcDNA3-CFP-CITED1 9S>E with or without hPTH(1-34) treatment (all P<0.05); these changes were not observed using CITED1 9S>A. Cells exposed to intermittent treatment with hPTH(1-34) expressed more ALP2, Runx2 and osteocalcin than vehicle-treated cells. These effects of hPTH(1-34) were inhibited in cells transfected with pcDNA3-CFP-CITED1 or pcDNA3-CFP-CITED1 9S>E, but were slightly enhanced by the alanine mutants. PKC activator (TPA) increased nuclear translocation of CITED1, whereas a PKC inhibitor (Go6983) blunted the effect of hPTH(1-34) on the nuclear translocation of wildtype CITED1 but not of CITED1 S(79)>E. The data indicated that serine phosphorylation at position 79 in the 63-84 domain is associated with PKC activation, and is required for both CITED1 nuclear translocation induced by PTH and the negative effects of CITED1 on osteoblastic differentiation and mineralization.

Activation of a non-cAMP/PKA signaling pathway downstream of the PTH/PTHrP receptor is essential for a sustained hypophosphatemic response to PTH infusion in male mice.

PTH increases urinary Pi excretion by reducing expression of two renal cotransporters [NaPi-IIa (Npt2a) and NaPi-IIc (Npt2c)]. In contrast to acute transporter regulation that is cAMP/protein kinase A dependent, long-term effects require phospholipase C (PLC) signaling by the PTH/PTHrP receptor (PPR). To determine whether the latter pathway regulates Pi through Npt2a and/or Npt2c, wild-type mice (Wt) and animals expressing a mutant PPR incapable of PLC activation (DD) were tested in the absence of one (Npt2a(-/-) or Npt2c(-/-)) or both phosphate transporters (2a/2c-dko). PTH infusion for 8 days caused a rapid and persistent decrease in serum Pi in Wt mice, whereas serum Pi in DD mice fell only transiently for the first 2 days. Consistent with these findings, fractional Pi excretion index was increased initially in both animals, but this increase persisted only when the PPR Wt was present. The hypophosphatemic response to PTH infusion was impaired only slightly in PPR Wt/Npt2c(-/-) or DD/Npt2c(-/-) mice. Despite lower baselines, PTH infusion in PPR Wt/Npt2a(-/-) mice decreased serum Pi further, an effect that was attenuated in DD/Npt2a(-/-) mice. Continuous PTH had no effect on serum Pi in 2a/2c-dko mice. PTH administration increased serum 1,25 dihydroxyvitamin D3 levels in Wt and DD mice and increased levels above the elevated baseline with ablation of either but not of both transporters. Continuous PTH elevated serum fibroblast growth factor 23 and blood Ca(2+) equivalently in all groups of mice. Our data indicate that PLC signaling at the PPR contributes to the long-term effect of PTH on Pi homeostasis but not to the regulation of 1,25 dihydroxyvitamin D3, fibroblast growth factor 23, or blood Ca(2+).

Loss of wnt/ß-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 ß-catenin from osteoblasts would cause bone marrow adiposity. Here, we determined whether postnatal deletion of ß-catenin in preosteoblasts, through conditional cre expression driven by the osterix promoter, causes bone marrow adiposity. Postnatal disruption of ß-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/ß-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 ß-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/ß-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.

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.

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.

Suppression of Wnt signaling by Dkk1 attenuates PTH-mediated stromal cell response and new bone formation.

Parathyroid hormone (PTH) suppresses Dickkopf 1 (Dkk1) expression in osteoblasts. To determine whether this suppression is essential for PTH-mediated Wnt signaling and bone formation, we examined mice that overexpress Dkk1 in osteoblasts (Dkk1 mice). Dkk1 mice were osteopenic due to abnormal osteoblast and osteoclast activity. When fed a low-calcium diet, and in two other models of hyperparathyroidism, these mice failed to develop the peritrabecular stromal cell response ("osteitis fibrosis") and new bone formation seen in wild-type mice. Despite these effects of Dkk1 overexpression, PTH still activated Wnt signaling in Dkk1 mice and in osteoblastic cells cultured from these mice. In cultured MC3T3E1 preosteoblastic cells, PTH dramatically suppressed Dkk1 expression, induced PKA-mediated phosphorylation of beta-catenin, and significantly enhanced Lef1 expression. Our findings indicate that the full actions of PTH require intact Wnt signaling but that PTH can activate the Wnt pathway despite overexpression of Dkk1.

Functional analysis of a type 1 parathyroid hormone receptor intracellular tail mutant [KRK(484-6)AAA]: effects on second messenger generation and cellular targeting.

The parathyroid hormone receptor type 1 (PTHR1) is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP) and primarily signals via intracellular pathways involving adenylyl cyclase and phospholipase C. The intracellular tail domain of the PTHR1 contributes to G protein subunit coupling that is important for second messenger signalling. In addition, the intracellular domain has a potential nuclear localization sequence (NLS) that, if functional, could point to an intracrine role for the receptor. In the present study, we have utilized 2 sets of constructs that employ either a [KRK(484-486)AAA](3Ala) mutation in the putative NLS or the non-mutant counterpart and included (a) the full-length rat PTHR1 with FLAG and c-myc epitope tags at the N-terminus and C-terminus, respectively (designated as PTHR1(3Ala)-TAG and PTHR1-TAG); and (b) only the putative NLS-containing intracellular domain (471-488), with green fluorescent protein (GFP) fused to the C-terminus (designated as GFP-(3Ala)471-488 or GFP-471-488). Porcine kidney LLC-PK1 cells stably expressing the PTHR1(3Ala)-TAG exhibited reduced signalling via both cAMP and cytosolic calcium transients in spite of greater cell surface expression relative to cells expressing PTHR1-TAG. We also examined the ability of the intracellular tail to influence the cellular localization of a heterologous protein. LLC-PK1 cells transiently transfected with GFP-471-488, exhibited increased fluorescence within the nucleus, relative to cells transfected with GFP alone that was not observed when cells were transiently transfected with the mutated construct, GFP-(3Ala)471-488. However, LLC-PK1 cells transiently transfected with either the full-length PTHR1-TAG or the PTHR1(3Ala)-TAG constructs did not exhibit nuclear localization of these receptors. Moreover, mouse osteoblast-like cells (MC3T3-E1) transiently expressing PTHR1-TAG also failed to demonstrate nuclear localization, although both full-length PTHR1 constructs exhibited plasma membrane immunofluorescence in both cell lines. Thus, the 484-486 sequence is critical for the full signalling responsiveness of the intact PTHR1, but the putative nuclear localization signal may not function as such within the intact receptor.

CBP/p300-interacting protein CITED1 modulates parathyroid hormone regulation of osteoblastic differentiation.

PTH regulates osteoblastic differentiation and activity and exerts different overall skeletal effects in vivo, depending on the schedule and dose of administration. In clonal Wt9 murine osteoblastic cells, mRNA and protein levels of CITED1 transcriptional coactivator were strongly up-regulated by human (h) PTH(1-34). Stimulation of CITED1 mRNA by PTH was transient, peaking at 4 h, concentration dependent, and blocked by actinomycin D but not cycloheximide. The stimulation was mimicked by forskolin, phorbol ester, and the cAMP-selective PTH analog [G(1),R(19)] hPTH (1-28) and inhibited completely by the protein kinase A inhibitor, H89 and partially by phorbol ester-induced protein kinase C depletion. Increased CITED1 expression was not maintained during persistent (24 h) PTH exposure. Cultured primary calvarial osteoblasts from neonatal homozygous or hemizygous CITED1-knockout (KO) mice achieved 2-fold greater mineralized nodule formation in comparison with wild type (WT) osteoblasts. This effect was blocked by restoration of CITED1 expression via adenoviral gene transfer. Intermittent administration of hPTH(1-34) (10 nm, for 4 h every 48 h) for 3-6 wk increased mineralization up to 2-fold over basal levels in both WT and CITED1 KO mouse calvarial cell cultures. Whereas the cAMP-selective [G(1),R(19)]hPTH(1-28) analog [at 100 nm, equivalent to 10 nm hPTH(1-34)] did not stimulate mineralization in WT cultures, it was twice as effective as hPTH(1-34) in CITED1 KO cultures. Thus, CITED1 negatively regulates osteoblastic differentiation in vitro and inhibits the cAMP-dependent stimulation of differentiation by intermittent PTH. We conclude also that PTH receptor signaling pathways independent of cAMP restrain osteoblastic differentiation, an effect normally obscured in the presence of CITED1 but revealed in its absence.

Expression of PTH1R constructs in LLC-PK1 cells: protein nuclear targeting is mediated by the PTH1R NLS.

This study demonstrates that the PTH1R NLS can target a fusion protein to the nucleus, and that this is blocked by sequences downstream of the NLS. GFP fused to the NLS showed a significant increase in nuclear targeting compared to GFP alone or GFP fused to a peptide of the same length. In previous studies, we demonstrated that the type I PTH/PTHrP receptor (PTH1R) localizes to the nucleus of cells within rat liver, kidney, uterus, ovary and gut. Similarly, nuclear localization of the PTH1R was observed in the cultured osteoblast-like cells MC3T3-E1, UMR106, ROS 17/2.8 and SaOS-2. We have identified a putative bipartite nuclear localization signal (NLS), from residues 471-488 in the protein sequence of the PTH1R. In this study, several PTH1R constructs were made in the Enhanced Green Fluorescent Protein (EGFP) expression vector (Clontech), transiently transfected into LLC-PK1 Clone 46 cells, and the resultant fusion protein expression followed by fluorescence microscopy. This particular clone of LLC-PK1 shows no biochemical response in vitro to parathyroid hormone. Constructs included the entire PTH1R sequence (PTH1R-GFP), the putative NLS fused to the C-terminus of GFP (GFP-NLS) or the NLS through to the C-terminus of the PTH1R fused to GFP (GFP-NLSCT). Deconvolution fluorescence microscopy of cells transfected with PTH1R-GFP showed abundant fluorescent signal throughout the cells with distinctly fluorescing plasma membranes. These cells also exhibited an increase in cAMP production in response to (0-10(-8) M) hPTH(1-34), with an increase in cAMP from 11 fmol/mug of protein to 101 fmol/microg. In contrast, cells transfected with the GFP-NLS construct showed significant nuclear sequestration of fluorescence as compared to GFP alone, GFP-NLSCT, or a short amino acid sequence fused to GFP (GFP-FFVAIYCFCNGEVQAEI). These results indicate that the NLS at residues 471-488 of the mature rat PTH1R is functional and plays a role in targeting the PTH1R the nucleus, also the addition of GFP to the C-terminus of the PTH1R still allows cAMP generation which will be useful for further studies.

Synthesis and characterization of novel biotinylated carboxyl-terminal parathyroid hormone peptides that specifically crosslink to the CPTH-receptor.

Parathyroid hormone (PTH) regulates calcium, phosphorous and skeletal homeostasis via interaction with the G protein-coupled PTH/PTHrP receptor, which is fully activated by the amino-terminal 34 amino-acid portion of the hormone. Recent evidence points to the existence of another class of receptors for PTH that recognize the carboxyl (C)-terminal region of intact PTH (1-84) (CPTHRs) and are highly expressed by osteocytes. Here we report the synthesis and characterization of two novel bifunctional CPTH ligands that include benzoylphenylalanine (Bpa) substitutions near their amino-termini and carboxyl-terminal biotin moieties, as well as a tyrosine(34) substitution to enable radioiodination. These peptides are shown to bind to CPTHRs with affinity similar to that of PTH (1-84) and to be specifically and covalently crosslinked to CPTHRs upon exposure to ultraviolet light. Crosslinking to osteocytes or osteoblastic cells generates complexes of 80 and 220 kDa, of which the larger form represents an aggregate that can be resolved into the 80 kDa. The crosslinked products can be further purified using immunoaffinity and avidin-based affinity procedures. While the molecular structure of the CPTHR(s) remains undefined, these bifunctional ligands represent powerful new tools for use in isolating and characterizing CPTHR protein(s).

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

UNLABELLED: 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. INTRODUCTION: Fibroblast growth factor 23 (FGF-23) is a novel phosphaturic factor discovered through genetic studies of patients with renal phosphate wasting disorders. Ablation of the FGF-23 gene in mice reduces renal phosphate excretion and increases serum phosphate, suggesting that FGF-23 is critical for normal phosphate homeostasis. We examined the role of dietary phosphate in the regulation of FGF-23 in humans. MATERIALS AND METHODS: Sixty-six healthy males and females were randomized to either phosphate-depleted or -loaded diets for 5 days, after a 4-day run-in diet. FGF-23 was measured using an "intact" assay that only detects intact FGF-23 peptide and with a "C-terminal" assay that measures both intact FGF-23 peptide and presumed biologically inactive carboxyl terminal fragments. The main outcome was the within group change in FGF-23 with either phosphate depletion or loading. RESULTS: Using the intact FGF-23 assay, mean FGF-23 area under the curve (AUC) decreased by 9 +/- 16% with phosphate depletion (p = 0.0041) and increased by 35 +/- 29% with loading (p < 0.0001). Using the C-terminal FGF-23 assay, mean FGF-23 AUC decreased by 8 +/- 12% with phosphate depletion (p = 0.0003) and increased by 13 +/- 20% with loading (p = 0.0016). Increases in FGF-23 with phosphate loading were greater with the intact assay than with the C-terminal assay (p = 0.0003). Using the intact assay only, FGF-23 was significantly associated with serum phosphate (r = 0.39, p < 0.01), 24-h urinary phosphate (r = 0.47, p < 0.01), fractional excretion of phosphate (r = 0.29, p < 0.01), and 1,25-dihydroxyvitamin D (r = -0.30, p < 0.01). The association between the assays was weak (r = 0.26, p < 0.01). CONCLUSIONS: Dietary phosphate is a key regulator of circulating FGF-23 levels in humans. Additionally, choice of assay is critical when performing physiologic investigations of FGF-23.

PTH/PTHrP receptor delays chondrocyte hypertrophy via both Runx2-dependent and -independent pathways.

The transcription factor, Runx2, promotes chondrocyte hypertrophy, whereas parathyroid hormone-related protein (PTHrP) delays this process. To examine whether PTHrP suppresses chondrocyte hypertrophy via Runx2-dependent or -independent pathways, Runx2 expression and chondrocyte differentiation were analyzed using bones from embryonic limbs of wild type and Runx2(-/-) mice. Treatment of cultured rudiments with PTH dramatically suppresses Runx2 mRNA levels in hypertrophic chondrocytes. PTH-induced delay of chondrocyte hypertrophy was observed in cultured tibiae from both Runx2(-/-) and wild-type embryos. This delay was also seen after PTH administration to limbs from wild type and Runx2(-/-) mice expressing Runx2 in chondrocytes via a collagen 2 promoter-driven transgene. To further explore Runx2-dependent and -independent effects of PTHrP, we examined embryonic tibiae and femurs from littermates null for PTHrP, Runx2, or both genes. Runx2(-/-) femurs exhibited no vascular invasion or chondrocytes expressing collagen type X or osteopontin mRNA. In contrast, Runx2(-/-)/PTHrP(-/-) mice exhibited limited vascular invasion and some chondrocytes expressing collagen X or osteopontin mRNA. In both tibia and femur, Runx2(-/-)/PTHrP(-/-) mice exhibited expanded regions of proliferating chondrocytes when compared to the same regions in PTHrP(-/-) mice. These data indicate that the delayed hypertrophy induced by PTHrP is mediated by both Runx2-dependent and -independent mechanisms.

Parathyroid hormone activates PKC-delta and regulates osteoblastic differentiation via a PLC-independent pathway.

PTH exerts major effects upon bone by activating PTH/PTHrP receptors (PTH1Rs) expressed on osteoblasts. The PTH1R is capable of engaging multiple signaling pathways in parallel, including Gs/adenylyl cyclase (AC), Gq/phospholipase C/protein kinase C (PLC/PKC) and a distinct mechanism, involving activation of PKC via a PLC-independent pathway, that depends upon ligand determinants within the PTH(29-34) sequence. The involvement of PLC-dependent vs. PLC-independent PKC activation in PTH action was studied in clonal PTH1R-expressing murine calvarial osteoblasts ("Wt9") using two signal-selective analogs, [G1,R19]hPTH(1-28) and [G1,R19]hPTH(1-34). Both analogs lack PLC signaling but differ in their capacity to activate the PLC-independent PKC pathway. Both hPTH(1-34) and [G1,R19]hPTH(1-34), but not [G1,R19]hPTH(1-28), increased differentiation of Wt9 cells during a 16-day alternate-daily treatment protocol. Wt9 cells expressed PKC-betaI, -delta, -epsilon and -zeta, none of which exhibited net translocation to membranes in response to hPTH(1-34) or either analog. hPTH(1-34) induced activation of membrane-associated PKC-delta, however, and a time- and concentration-dependent increase in cytosolic [phospho-Thr505]PKC-delta which was maximal within 40 s at 100 nM in both Wt9 cells and primary osteoblasts. This response was mimicked by [G1,R19]hPTH(1-34) but not by [G1,R19]hPTH(1-28). Increased expression of bone sialoprotein (BSP) and osteocalcin (OC) mRNAs induced by PTH(1-34) and [G1,R19]hPTH(1-34) in Wt9 cells was blocked by rottlerin, a PKC-delta inhibitor. We conclude that PTH1Rs activate PKC-delta by a PLC-independent, PTH(29-34)-dependent mechanism that promotes osteoblastic differentiation.

Parathyroid hormone secretion and action: evidence for discrete receptors for the carboxyl-terminal region and related biological actions of carboxyl- terminal ligands.

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.

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 degrees 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.

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.

Estrogen inhibition of PTH-stimulated osteoclast formation and attachment in vitro: involvement of both PKA and PKC.

Estrogens modulate the catabolic effects of PTH on bone in vivo and in vitro. PTH-stimulated cAMP accumulation in osteoblasts is thought to be linked to increased osteoclastic activity, but the precise mechanism is still unknown. In cocultures of clonal marrow stromal cells (MS1) and normal mouse spleen cells, both 1,25-dihydroxyvitamin D3 and rat PTH (rPTH)-(1-34) can induce the formation of tartrate-resistant acid phosphatase- and calcitonin receptor-positive multinucleated osteoclast-like cells, which can attach to dentine slices and produce resorption pits. In this system, osteoclastogenesis stimulated by PTH, but not by 1,25-dihydroxyvitamin D3, was suppressed by 17beta-E2 (10(-10)-10(-8) M), whereas 17alpha-E2 (10(-8) M) had no effect. Exposure to 10(-8) M 17beta-E2, but not 17alpha-E2, also significantly decreased the PTH-induced attachment of osteoclast-like cells to dentine slices. 17beta-E2 inhibited osteoclast-like cell formation induced by 8-bromo-cAMP (10(-4) M), 12-O-tetradecanoylphorbol 13-acetate (10(-8) M), or rat PTH-(1-34) (10(-7) M) in combination with either rp-adenosine-3',5'-cyclic monophosphorothioate (10(-4) M) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (10(-5) M). 17beta-E2 suppressed the partial stimulation of tartrate-resistant acid phosphatase-positive multinucleated osteoclast-like cell formation induced by [Arg(2)]human (h) PTH-(1-34) (10(-7) M) or hPTH-(3-34) (10(-7) M), but not that caused by 10(-7) M hPTH-(53-84). We conclude that estrogens suppress PTH-stimulated osteoclast-like cell formation by blocking both the cAMP-dependent PKA pathway and the PLC-coupled calcium/PKC pathway. In addition to inhibiting formation of osteoclasts and promoting their apoptosis, estrogen may regulate bone resorption by blocking attachment of osteoclasts to bone.