Osteoblastic cells regulate the haematopoietic stem cell niche.

Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. 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). Here we show that PPR-stimulated 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, ligand-dependent 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 gamma-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.

Humoral hypercalcemia of malignancy. Release of a prostaglandin-stimulating bone-resorbing factor in vitro by human transitional-cell carcinoma cells.

Secretion by tumor cells of circulating bone-resorbing factors may frequently underlie the hypercalcemia that occurs in patients with malignancy. Efforts to identify the responsible mediators have been hampered by a lack of available human tumor cell systems suitable for study of the pathogenesis of the humoral hypercalcemia syndrome. We have established a transitional-cell carcinoma (TCC) line in vitro from a patient with humoral hypercalcemia. These cells are tumorigenic and cause hypercalcemia in athymic nude mice. Culture medium conditioned by TCC cells contains potent bone-resorbing activity in vitro, the physical and biological properties of which are similar to those of bone-resorbing activity present in the original patient's urine. The bone-resorbing activity of the TCC factor is accompanied by increased prostaglandin release from bone and is blocked by indomethacin and calcitonin. The TCC-derived bone-resorbing activity coelutes with prostaglandin-stimulating activity during gel filtration with an approximate molecular weight of 15,000. This activity is nondialyzable, stable to concentrated urea and reducing agents, and destroyed by boiling. The TCC factor does not increase cyclic AMP production in bone or kidney bioassays and does not exhibit transforming growth factor activity. We conclude that a unique macromolecular factor released by TCC cells causes bone resorption by a mechanism dependent upon stimulation of bone cell cyclooxygenase, and that this factor is the probable cause of the hypercalcemia in vivo. The TCC cell line provides a new model for study of the human humoral hypercalcemia syndrome.

Contributions of parathyroid hormone (PTH)/PTH-related peptide receptor signaling pathways to the anabolic effect of PTH on bone.

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 PLC-independent 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 microg/kg), [G(1),R(19)]hPTH(1-34) (120 microg/kg), and [G(1),R(19)]hPTH(1-28) (800 microg/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 that 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.

Receptors specific for the carboxyl-terminal region of parathyroid hormone on bone-derived cells: determinants of ligand binding and bioactivity.

PTH comprises 84 amino acids of which the first 34 are sufficient for full activation of the classical PTH/PTHrP receptor, the type 1 PTH receptor. It is known that multiple carboxyl (C)-terminal fragments of PTH are present in the blood and that they comprise the majority of circulating PTH. C-PTH fragments, previously regarded as by-products of PTH metabolism, are directly secreted by the parathyroid glands or arise from the peripheral cleavage of the intact hormone. Compelling evidence now strongly suggests that these C-PTH fragments mediate biological effects via activation of a receptor that specifically recognizes the C-terminal portion of intact PTH, and this receptor is therefore named the carboxyl-terminal PTH receptor (CPTHR). We have previously reported that osteocytes abundantly express this novel receptor and that its activation is involved in cell survival and communication. Here we report the characterization of determinants of PTH that are required for high-affinity binding to the CPTHR. Using synthetic PTH peptides harboring alanine substitution or truncations, we showed the existence of discrete binding domains and critical residues within the intact hormone. We have furthermore identified eight amino acids within the PTH sequence that play key roles in optimizing the binding affinity of C-PTH fragments to CPTHRs. These include the tripeptide sequence Arg(25)-Lys(26)-Lys(27), the dibasic sequence Lys(53)-Lys(54), and three additional residues within the PTH (55-84) sequence, Asn(57), Lys(65), and Lys(72). Functional analysis of these residues demonstrated a strong correlation between binding affinity and biological effect and points to a potential role of CPTHR activation in regulating bone cell survival.

Inhibition of parathyroid hormone responsiveness in clonal osteoblastic cells expressing a mutant form of 3',5'-cyclic adenosine monophosphate-dependent protein kinase.

PTH activates multiple acute intracellular signals within responsive target cells, but the importance of cAMP vs. other second messenger signals in mediating different biological responses to PTH is not known. To address these questions, we developed a genetic approach to block activation of the cAMP-dependent protein kinase (PK-A) in PTH-responsive cell lines. Clonal rat osteosarcoma cells (UMR 106-01) were stably transfected with REV-I, a plasmid that directs synthesis of a mutant cAMP-resistant form of the type I regulatory subunit of PK-A. In the transfected bone cells, most of the catalytic subunits of PK-A were associated with the mutant regulatory subunit, and activation of PK-A by cAMP was correspondingly inhibited. We have characterized one such mutant (UMR 4-7) that expressed large amounts of mutant mRNA and exhibited inducible blockade of PK-A via the REV-1 metallothionein promoter. In the absence of metallothionein induction, these cells exhibited nearly normal PTH responsiveness, but after REV-1 induction by Zn2+, they were resistant to PTH-induced activation of PK-A and regulation of membrane phospholipid synthesis by both PTH and cAMP analogs. The mutant UMR 4-7 cell provides a model system in which the consequences of cAMP production by PTH or other agonists that activate adenylate cyclase in osteoblasts may be specifically inhibited by brief exposure to Zn2+. Such mutant cell lines will facilitate further investigation of the linkage between early signalling events and subsequent biological responses in the action of PTH and other agonists on target cells in bone.

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 microM 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.

Properties of parathyroid hormone receptors on circulating bovine lymphocytes.

Binding of parathyroid hormone (PTH) to circulating bovine lymphocytes was studied using, as the radioligand, a synthetic sulfur-free analog of bovine PTH, [Nle8,Nle18,Tyr34]bPTH-(1-34)amide, which was labeled to high specific activity with 125I and was purified by reverse-phase high-performance liquid chromatography. Binding of PTH to lymphocytes satisfies several criteria indicative of a specific interaction between the hormone and its receptor. Specific binding is saturable at 3.3 fmoles of radioligand bound per 10(7) cells, occurs more rapidly at 37 degrees C than at lower temperatures, and reaches equilibrium within 2 hr at 15 degrees C. Inhibition of specific binding occurs with intact PTH, with biologically active PTH analog or fragment, and with synthetic PTH antagonists, but not with biologically inactive PTH fragments, or peptide hormones unrelated to PTH antagonists, but not with biologically inactive PTH fragments, or peptide hormones receptors on lymphocytes and those previously reported with receptors in canine renal membranes, and on rat osteosarcoma cells. The dissociation constant (Kd) is approximately 10(-9) M, as calculated from the association and dissociation rate constants. This correlates very closely both with the apparent Kd, as estimated from Scatchard analysis of radioligand saturation and competition studies, and with previously reported Kd of PTH receptors in canine renal membranes and on intact rat osteosarcoma and opossum kidney cells. In addition, the relative binding affinity of intact hormone and a synthetic PTH agonist to to receptors on lymphocytes correlates closely with the relative biologic potency of these peptides in stimulating adenylate cyclase in membranes from these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Conditionally immortalized murine osteoblasts lacking the type 1 PTH/PTHrP receptor.

Osteoblasts synthesize and mineralize bone matrix and are principal target cells for parathyroid hormone (PTH). The type 1 PTH/PTH-related protein (PTHrP) receptor (PTH1R), cloned from rat osteoblastic cells, activates multiple intracellular signaling mechanisms. The specific roles of these PTH1R signals, or of responses to other types of PTH receptors that may be expressed, in regulating osteoblast function are incompletely understood. Use of established mammalian osteoblastic cell lines has led to much understanding of PTH action in bone, although such cells are of neoplastic origin or have other characteristics that compromise their validity as models of normal osteoblasts. To examine the role of the PTH1R in osteoblast biology, we have isolated a series of clonal murine calvarial osteoblastic cell lines that are only conditionally immortalized, via expression of a transgene encoding the tsA58 temperature-sensitive SV40 large T antigen, and that lack both functional alleles of the PTH1R gene. When cultured under nontransforming conditions, these cells stopped proliferating, expressed a series of characteristic osteoblastic genes (including the nonfunctional remnant of the PTH1R gene), and, after 3-4 weeks, produced mineralized bone nodules in a manner that was regulated by 1,25-dihydroxyvitamin D3 but not by PTH(1-84). Cyclic AMP measurements revealed no evidence of expression of alternate species of Gs-linked PTH receptors. Stable transfection with PTH1R cDNA reconstituted both PTH binding and adenylyl cyclase activation, increased basal osteocalcin expression, and supported PTH stimulation of c-Fos expression and matrix mineralization. These conditionally transformed, PTH1R(-/-) clonal osteoblastic cell lines should prove useful for studies of the regulation of osteoblast differentiation and function by both endogenous nonclassical species of PTH (or PTHrP) receptors and mutant signal-selective PTH1Rs.

Stimulation of protein kinase C activity in cells expressing human parathyroid hormone receptors by C- and N-terminally truncated fragments of parathyroid hormone 1-34.

The parathyroid hormone (PTH) fragment PTH(1-34) stimulates adenylyl cyclase, phospholipase C (PLC), and protein kinase C's (PKCs) in cells that express human, opossum, or rodent type 1 PTH/PTH-related protein (PTHrP) receptors (PTHR1s). Certain carboxyl (C)-terminally truncated fragments of PTH(1-34), such as human PTH(1-31) [hPTH-(1-31)NH2], stimulate adenylyl cyclase but not PKCs in rat osteoblasts or PLC and PKCs in mouse kidney cells. The hPTH(1-31)NH2 peptide does fully stimulate PLC in HKRK B7 porcine renal epithelial cells that express 950,000 transfected hPTHR1s per cell. Amino (N)-terminally truncated fragments, such as bovine PTH(3-34) [bPTH(3-34)], hPTH(3-34)NH2, and hPTH(13-34), stimulate PKCs in Chinese hamster ovary (CHO) cells expressing transfected rat receptors, opossum kidney cells, and rat osteoblasts, but an intact N terminus is needed to stimulate PLC via human PTHR1s in HKRK B7 cells. We now report that the N-terminally truncated analogs bPTH(3-34)NH2 and hPTH(13-34)OH do activate PKC via human PTHR1s in HKRK B7 cells, although less effectively than hPTH(1-34)NH2 and hPTH(1-31)NH2. Moreover, in a homologous human cell system (normal foreskin fibroblasts), these N-terminally truncated fragments stimulate PKC activity as strongly as hPTH(1-34)NH2 and hPTH(1-31)NH2. Thus, it appears that unlike their opossum and rodent equivalents, hPTHR1s can stimulate both PLC and PKCs when activated by C-terminally truncated fragments of PTH(1-34). Furthermore, hPTHR1s, like the PTHR1s in rat osteoblasts, opossum kidney cells, and rat PTHR1-transfected CHO cells also can stimulate PKC activity by a mechanism that is independent of PLC. The efficiency with which the N-terminally truncated PTH peptides stimulate PKC activity depends on the cellular context in which the PTHR1s are expressed.

Lactam formation increases receptor binding, adenylyl cyclase stimulation and bone growth stimulation by human parathyroid hormone (hPTH)(1-28)NH2.

Human parathyroid hormone (1-28)NH2 [hPTH(1-28)NH2] is the smallest of the PTH fragments that can fully stimulate adenylyl cyclase in ROS 17/2 rat osteoblast-like osteosarcoma cells. This fragment has an IC50 of 110 nM for displacing 125I-[Nle8,18,Tyr34]bovine PTH(1-34)NH2 from HKRK B7 porcine kidney cells, which stably express 950,000 human type 1 PTH/PTH-related protein (PTHrP) receptors (PTH1Rs) per cell. It also has an EC50 of 23.9 nM for stimulating adenylyl cyclase in ROS 17/2 cells. Increasing the amphiphilicity of the alpha-helix in the residue 17-28 region by replacing Lys27 with Leu and stabilizing the helix by forming a lactam between Glu22 and Lys26 to produce the [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 analog dramatically reduced the IC50 for displacing 125I-[Nle8,18,Tyr34]bPTH(1-34)NH2 from hPTH1Rs from 110 to 6 nM and dropped the EC50 for adenylyl cyclase stimulation in ROS 17/2 cells from 23.9 to 9.6 nM. These modifications also increased the osteogenic potency of hPTH(1-28)NH2. Thus, hPTH(1-28)NH2 did not significantly stimulate either femoral or vertebral trabecular bone growth in rats when injected daily at a dose of 5 nmol/100 g body weight for 6 weeks, beginning 2 weeks after ovariectomy (OVX), but it strongly stimulated the growth of trabeculae in the cancellous bone of the distal femurs and L5 vertebrae when injected at 25 nmol/100 g body weight. By contrast [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 significantly stimulated trabecular bone growth when injected at 5 nmol/100 g of body weight. Thus, these modifications have brought the bone anabolic potency of hPTH(1-28)NH2 considerably closer to the potencies of the larger PTH peptides and analogs.

Bone collagen synthesis in vitro: structure/activity relations among parathyroid hormone fragments and analogs.

The structural requirements for the inhibition of net bone collagen synthesis by parathyroid hormone (PTH) in vitro have been examined by study of the effects of selected fragments and analogs of bovine PTH (bPTH) upon the incorporation of [3H]proline into collagenase-digestible and -nondigestible proteins by neonatal mouse calvarial bone in organ culture. At concentrations of 10(-10)-10(-7) M, the amino-terminal fragment bPTH-(1-34) was found to be as potent as intact bPTH in the specific suppression of net bone collagen synthesis after 24 h in culture. The synthetic fragments bPTH-(1-30), bPTH-(1-28), and bPTH-(3-34) were approximately 3%, 1%, and 0.2% as active, respectively, as bPTH-(1-34), in good agreement with previous estimates of the relative potencies of these hormonal fragments on bone resorption in vitro and in vivo and on adenylate cyclase activation in and receptor binding to isolated renal membranes. The amino-terminal analog [Ser1]bPTH-(1-34) displayed no reduction in biological activity compared with bPTH-(1-34), as previously found for bone resorption in vivo. The overall results with this assay system indicate a minimum sequence for biological activity that extends from residues 3-28 of intact bPTH, which is consistent with similar estimates in other test systems and emphasizes the importance of the aminoterminus of the hormone in the expression of its biological effects on bone formation as well as resorption. Moreover, these findings support the potential usefulness of the mouse calvarial culture system in predicting the skeletal activity in vivo of new synthetic analogs of PTH.

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 concentrations (EC50 = 10 nM vs. 1 nM). Surprisingly, hPTH(1-31) fully stimulated phospholipase C (PLC), via both species of PTH1Rs, with potency that was similar (hPTH1Rs) or slightly reduced (rat PTH1Rs), relative to that of hPTH(1-34). hPTH(1-30), however, was 5-fold less potent than hPTH(1-34) in activating PLC via hPTH1Rs and showed weak and only partial activity via the rat PTH1R. Comparable results were obtained when human and rat PTH1Rs were transiently expressed heterologously in COS-7 cells or homologously in HEK 293 and UMR 106-01 cells, respectively. Binding affinities of these C-truncated peptides to human and rat PTH1Rs were concordant with their relative potencies in activating PLC. We conclude that hPTH(1-31) and, to a lesser extent, hPTH(1-30) can activate PLC, as well as AC, via both rat and human PTH1Rs. Accordingly, a role for PLC activation in the anabolic action of PTH in vivo cannot be excluded.

Forskolin-induced homologous desensitization via an adenosine 3',5'-monophosphate-dependent mechanism(s) in human osteoblast-like SaOS-2 cells.

We have reported that pretreatment of human SaOS-2 osteoblast-like cells with forskolin (Fsk; 10(-5) M) for 4 h strikingly inhibited subsequent cAMP responsiveness to a second challenge with Fsk (Fsk-induced homologous desensitization) without altering the responses to PTH or vasoactive intestinal peptide (VIP). Pretreatment with PTH acutely augmented Fsk responsiveness, despite desensitizing the cells to rechallenge with PTH. The present studies were performed to investigate the mechanism of this differential desensitization. Fsk-induced desensitization was not mimicked by 1,9-dideoxyforskolin, a Fsk analog that does not activate adenylate cyclase (AC) but does reproduce certain cAMP-independent effects of Fsk. Fsk-induced homologous desensitization was also completely blocked in a cAMP-resistant mutant SaOS-2 cell line (Ca 4A), in which protein kinase-A (PKA) is not activated by endogenous cAMP. However, pretreatment with PTH (or VIP), which induced a large increase in cAMP, did not attenuate, but, rather, increased, the subsequent cAMP response to Fsk. Potentiation by PTH was also observed in Ca 4A cells. Pretreatment of SaOS-2 cells with pertussis toxin (100 ng/ml) for 12 h strikingly inhibited the initial cAMP response to Fsk, although Fsk-induced homologous desensitization was still clearly observed. Pretreatment with cholera toxin (1 microgram/ml) completely prevented Fsk-induced homologous desensitization. Combinations of maximal concentrations of Fsk plus hormones such as human PTH, human PTH-related peptide, or VIP elicited cAMP responses that were much more than additive, an effect not observed with combinations of hormones alone. We conclude that 1) Fsk-induced homologous desensitization of the AC response of SaOS-2 cells to a second challenge with Fsk is dependent upon activation of PKA; 2) one or more pertussis toxin-sensitive G-proteins contribute to full AC activation by Fsk, but are not involved in homologous desensitization; 3) augmentation by PTH (or VIP) pretreatment of Fsk-dependent AC activation involves an effector(s) other than PKA. These results provide further evidence that the regulation of AC responsiveness in SaOS-2 cells by PTH or VIP is complex and cannot be explained by activation of PKA alone.

Mechanisms of desensitization to parathyroid hormone in human osteoblast-like SaOS-2 cells.

Signal transduction by the PTH receptor is now known to involve generation of multiple second messengers. Desensitization of the adenylate cyclase response to PTH is a common feature of bone- and kidney-derived target cells; however, no single mechanism appears to explain desensitization in the different cell types studied. To examine the role of protein kinase-A (PKA) in homologous desensitization to PTH, we employed human SaOS-2 osteoblast-like cells and a mutant subclone (Ca 4A), which expresses an inducible cAMP-resistant form of PKA. Pretreatment of SaOS-2 cells with PTH for 4 h reduced by 60-80% the cAMP response to subsequent rechallenge with the hormone. This homologous desensitization was significantly, but not completely, inhibited in Ca 4A cells. Desensitization was not mimicked by pretreatment of the cells with forskolin. PTH binding to its receptor was reduced 50% in both SaOS-2 and Ca 4A cells after 4-h incubation with PTH (homologous down-regulation), whereas forskolin did not cause receptor down-regulation. Pretreatment with the ionophore ionomycin for 4-24 h did not mimic desensitization to PTH. Both desensitization to PTH and receptor down-regulation were induced, however, by pretreatment with a phorbol ester (12-O-tetradecanoyl phorbol-13-acetate), and these effects were blocked completely by staurosporine. PTH-induced desensitization was not blocked by staurosporine, and receptor down-regulation was enhanced by the drug. Pertussis toxin did not prevent desensitization induced by either PTH or 12-O-tetradecanoyl phorbol-13-acetate. We conclude that homologous desensitization to PTH in SaOS-2 cells involves both cAMP-dependent and -independent mechanisms. Homologous PTH receptor down-regulation apparently is mediated by mechanisms independent of PKA activation. Neither pathway of homologous desensitization to PTH involves the action of pertussis toxin-sensitive G-proteins.

Human PTH-(7-84) inhibits bone resorption in vitro via actions independent of the type 1 PTH/PTHrP receptor.

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.

Regulation of 1,25-dihydroxyvitamin D3 receptors by parathyroid hormone in osteoblastic cells: role of second messenger pathways.

The regulation of vitamin D receptor (VDR) abundance in MC3T3-E1 mouse osteoblasts and UMR 106-01 rat osteosarcoma cells by rat PTH 1-34, human PTH-related protein 1-34, and agents that activate specific signal transduction pathways was studied. Treatment of these cells with forskolin (FSK) caused up-regulation of VDR, whereas treatment with phorbol esters suppressed VDR levels. PTH or PTH-related protein treatment induced a 2- to 3-fold increase in VDR, which was equivalent to that elicited by FSK in UMR 106-01 cells but less than the FSK-induced increase (approximately 8-fold) in MC3T3-E1 cells. PTH treatment of MC3T3-E1 cells resulted in an approximately 3-fold increase in VDR levels with maximum stimulation occurring at 10(-9) M PTH after 4 h of treatment. In UMR 4-7 cells, a subclone of UMR 106-01 cells that express cAMP resistance due to regulated expression of a mutant form of the type 1 regulatory subunit of the cAMP-dependent protein kinase A (PKA), the up-regulation of VDR abundance due to FSK and PTH treatment was mostly prevented. Pretreatment of MC3T3-E1 cells with staurosporine, an inhibitor of PKC, resulted in an approximately 3-fold increase in basal VDR levels but did not enhance the PTH-mediated up-regulation of VDR. Collectively, these data suggest that the increase in VDR abundance observed in these target cells is mainly due to the activation of the PKA signal transduction pathway. Treatment of UMR 106-01 cells with PTH for 4 h before exposure of the cells to 1,25-dihydroxyvitamin D3 resulted in a 2-fold increase in the induction of 25-hydroxyvitamin D3-24 hydroxylase messenger RNA. Thus, exposure of target cells to PTH augments their response to 1,25-dihydroxyvitamin D3 due to up-regulation of VDR abundance.

Signal-selectivity of parathyroid hormone (PTH)/PTH-related peptide receptor-mediated regulation of differentiation in conditionally immortalized growth-plate chondrocytes.

Type-1 PTH/PTH-related peptide receptors (PTH1Rs), which activate both adenylyl cyclase and phospholipase C (PLC), control endochondral bone development by regulating chondrocyte differentiation. To directly analyze PTH1R function in such cells, we isolated conditionally transformed clonal chondrocytic cell lines from tibial growth plates of neonatal mice heterozygous for PTH1R gene ablation. Among 104 cell lines isolated, messenger RNAs for PTH1R, collagen II, and collagen X were detected in 28%, 90%, and 29%, respectively. These cell lines were morphologically diverse. Some appeared large, rounded, and enveloped by abundant extracellular matrix; whereas others were smaller, flattened, and elongated. Two PTH1R-expressing clones showed similar PTH1R binding and cAMP responsiveness to PTH and PTH-related peptide but disparate morphologic features, characteristic of hypertrophic (hC1--5) or nonhypertrophic (nhC2--27) chondrocytes, respectively. hC1--5 cells expressed messenger RNAs for collagen II and X, alkaline phosphatase (ALP), and matrix GLA protein, whereas nhC2--27 cells expressed collagen II and Indian hedgehog but not collagen X or ALP. In hC1--5 cells, PTH and cAMP analog, but not phorbol ester, inhibited both ALP and mineralization. PTH1R-null hC1--5 subclones were isolated by in vitro selection and then reconstituted by stable transfection with wild-type PTH1Rs or mutant (DSEL) PTH1Rs defective in PLC activation. ALP and mineralization were inhibited similarly via both forms of the receptor. These results indicate that PLC activation is not required for PTH1R regulation of mineralization or ALP in hypertrophic chondrocytes and are consistent with a major role for cAMP in regulating differentiation of hypertrophic chondrocytes.

Receptors for the carboxyl-terminal region of pth(1-84) are highly expressed in osteocytic cells.

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.

Conditionally immortalized murine bone marrow stromal cells mediate parathyroid hormone-dependent osteoclastogenesis in vitro.

PTH recruits and activates osteoclasts to cause bone resorption. These actions of PTH are thought to be mediated indirectly via type 1 PTH/PTH-related peptide receptors (PTH1Rs) expressed by adjacent marrow stromal or osteoblastic cells, although some evidence suggests that PTH may act directly on early hematopoietic osteoclast progenitors. We have established clonal, conditionally immortalized, PTH-responsive, bone marrow stromal cell lines from mice that harbor both a transgene encoding a temperature-sensitive mutant of the simian virus 40 large T antigen and deletion of a single allele of the PTH1R gene. Of 60 stromal cell lines isolated, 45 expressed functional PTH1Rs. During coculture with normal murine spleen cells, 5 of 42 such cell lines could support formation of tartrate-resistant acid phosphatase-positive, multinucleated cells (TRAP+ MNCs) in response to 1,25-dihydroxyvitamin D3, but only 2 of these did so in response to PTH. One of these, MS1 cells, expressed numerous cytokines and proteins characteristic of the osteogenic lineage and showed increased production of interleukin-6 in response to PTH. MS1 cells supported dose-dependent induction by rat (r) PTH-(1-34) (0.1-100 nM) of TRAP+ MNCs that expressed calcitonin receptors and formed resorption lacunae on dentine slices. This effect of PTH, which required cell to cell contact between MS1 and spleen cells, was mimicked by coadministration of cAMP analog and phorbol ester but only partially by either agent alone. The carboxyl-terminal fragment rPTH-(53-84) also induced osteoclast-like cell formation, but the maximal effect was only 30% as great as that of rPTH-(1-34). Importantly, rPTH-(1-34) induced TRAP+ MNC formation even when PTH1R-/- osteoclast progenitors (from fetal liver of mice homozygous for ablation of the PTH1R gene) were cocultured with MS1 cells. We conclude that activation of PTH1Rs on cells of the osteoclast lineage is not required for PTH-(1-34)-induced osteoclast formation in the presence of appropriate PTH-responsive marrow stromal cells. MS1 cells provide a useful model for further study of PTH regulation of osteoclastogenesis.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent and cAMP-independent regulation of parathyroid hormone receptors on UMR 106-01 osteoblastic osteosarcoma cells.

The osteoblast-like cells, UMR 106-01, express PTH receptors that are coupled to adenylate cyclase. Recently, we reported the isolation of a UMR 106-01 subclone, UMR 4-7, that is stably transfected with a Zn(++)-inducible mutant of the regulatory subunit of protein kinase A. Incubation of UMR 4-7 cells with Zn++ renders the cells unresponsive to cAMP agonists. This subclone, therefore, seemed particularly suitable for studies of PTH receptor regulation. In UMR 106-01 cells, PTH receptors are strikingly down-regulated by pretreatment with 8-Br-cAMP or 3-isobutyl-1-methylxanthine for 2 days. In UMR 4-7 cells, this effect is totally prevented by prior and concurrent treatment with Zn++. Zn++ addition to UMR 106 cells does not modify these responses. Treatment with the PTH agonist [Nle8,18,Tyr34]bovine PTH(1-34)NH2 [(NlePTH(1-34)] also markedly down-regulates PTH receptors in UMR 106 cells, but this effect is only partially inhibited in Zn(++)-induced UMR 4-7 cells. At high doses, the PTH antagonist, [Nle8,18,Tyr34]bovine PTH(3-34)NH2 [NlePTH(3-34)] also (partially) reduces PTH receptor availability. Receptor regulation by NlePTH(3-34) is not blocked in the cAMP-resistant cells, however. Coincubation of submaximal doses of NlePTH(1-34) (1 nM) with NlePTH(3-34) (1 microM) reduces receptor availability more than when the cells are exposed to either ligand alone. This decrease is only partially inhibited in Zn(++)-induced UMR 4-7 cells. In contrast to its additive effect on receptor regulation, NlePTH(3-34) efficiently competes for binding to the PTH receptor in UMR 106-01 cells and antagonizes the stimulatory effects of NlePTH(1-34) on both intracellular cAMP accumulation and gene expression driven by a transiently transfected synthetic cAMP-responsive enhancer. In conclusion, homologous down-regulation of PTH receptors is mediated by activation of both cAMP-dependent (via protein kinase A) and cAMP-independent pathways. PTH activates both pathways, whereas the effect of NlePTH(3-34) appears to be exclusively cAMP-independent. These results give new insights into mechanisms of PTH receptor regulation.