Role of protein kinase A, phospholipase C and phospholipase D in parathyroid hormone receptor regulation of protein kinase Calpha and interleukin-6 in UMR-106 osteoblastic cells

Parathyroid hormone (PTH) stimulates both bone formation and resorption by activating diverse osteoblast signalling pathways. Upstream signalling for PTH stimulation of protein kinase C-alpha (PKCalpha) membrane translocation and subsequent expression of the pro-resorptive cytokine interleukin-6 (IL-6) was investigated in UMR-106 osteoblastic cells. PTH 1-34, PTH 3-34, PTHrP and PTH 1-31 stimulated PKCalpha translocation and IL-6 promoter activity. Pharmacologic intervention at the adenylyl cyclase (AC) pathway (forskolin, IBMX, PKI) failed to alter PTH 1-34- or PTH 3-34-stimulated PKCalpha translocation. The phosphoinositol-phospholipase C (PI-PLC) antagonist U73122 slightly decreased PTH 1-34-stimulated PKCalpha translocation; however, the control analogue U73343 acted similarly. Propranolol, an inhibitor of phosphatidic acid (PA) phosphohydrolase, decreased diacylglycerol (DAG) formation and attenuated PTH 1-34- and PTH 3-34-stimulated PKCalpha translocation and IL-6 promoter activity, suggesting a phospholipase D (PLD)-dependent mechanism. This is the first demonstration that PLD-mediated signalling leads to both PKC-alpha translocation and IL-6 promoter activation in osteoblastic cells.     

Distinct roles for mitogen-activated protein kinase phosphatase-1 (MKP-1) and ERK-MAPK in PTH1R signaling during osteoblast proliferation and differentiation

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) activate one single receptor (PTH1R) which mediates catabolic and anabolic actions in the bone. Activation of PTH1R modulates multiple intracellular signaling responses. We previously reported that PTH and PTHrP down-regulate pERK1/2 and cyclin D1 in differentiated osteoblasts. In this study we investigate the role of MAPK phosphatase-1 (MKP-1) in PTHrP regulation of ERK1/2 activity in relation to osteoblast proliferation, differentiation and bone formation. Here we show that PTHrP increases MKP-1 expression in differentiated osteoblastic MC3T3-E1 cells, primary cultures of differentiated bone marrow stromal cells (BMSCs) and calvarial osteoblasts. PTHrP had no effect on MKP-1 expression in proliferating osteoblastic cells. Overexpression of MKP-1 in MC-4 cells inhibited osteoblastic cell proliferation. Cell extracts from differentiated MC-4 cells treated with PTHrP inactivate/dephosphorylate pERK1/2 in vitro; immunodepletion of MKP-1 blocked the ability of the extract to dephosphorylate pERK1/2; these data indicate that MKP-1 is involved in PTHrP-induced pERK1/2 dephosphorylation in the differentiated osteoblastic cells. PTHrP regulation of MKP-1 expression is partially dependent on PKA and PKC pathways. Treatment of nude mice, bearing ectopic ossicles, with intermittent PTH for 3 weeks, up-regulated MKP-1 and osteocalcin, a bone formation marker, with an increase in bone formation. These data indicate that PTH and PTHrP increase MKP-1 expression in differentiated osteoblasts; and that MKP-1 induces growth arrest of osteoblasts, via inactivating pERK1/2 and down-regulating cyclin D1; and identify MKP-1 as a possible mediator of the anabolic actions of PTH1R in mature osteoblasts.     

Antiproliferative effect of the C-terminal fragments of parathyroid hormone-related protein,PTHrP-(107–111) and (107–139), on osteoblastic osteosarcoma cells

The C-terminal region of parathyroid hormone-related protein (PTHrP) containing the sequence (107–111) appears to be a potent inhibitor of osteoclastic bone resorption. In the present study, we have investigated the effect of human (h)PTHrP (107–139) and hPTHrP (107–111)NH₂ on the proliferation of osteoblastic rat osteosarcoma UMR 106 cells. We found that both C-terminal PTHrP peptides, like hPTHrP (1–141), were antimitogenic for these cells, between 1 pM and 10 nM. [Tyr³⁴]hPTHrP (1–34)NH₂ was as potent as these peptides but less effective as growth inhibitor in these cells. UMR 106 cells were found to produce and secrete immunoreactive PTHrP. Addition of anti-PTHrP neutralizing antibodies to C- and N-terminal epitopes of PTHrP increased the growth of these cells. Our data suggest that the antiproliferative effect of these C-terminal PTHrP analogs may be independent of cyclic adenosine 3′:5′-monophosphate (cAMP) and mediated by protein kinase C. These findings support an autocrine role of PTHrP in bone metabolism. J. Cell. Physiol. 170:209–215, 1997. © 1997 Wiley-Liss, Inc.     

Interleukin-4 as a potent inhibitor of bone resorption

A possible role of interleukin-4 (IL-4) in the regulation of bone turnover was assessed by employing a 45Ca prelabeled-fetal mouse long bone culture system. IL-4 inhibited the bone resorption stimulated by parathyroid hormone (PTH), PTH related protein (PTHrP), 1 alpha, 25, dihydroxy-vitamin D3 [1 alpha, 25 (OH)2 D3], interleukin-1 alpha and - 1 beta (IL-1 alpha, IL-1 beta) and prostaglandin E2 (PGE2). Anti-IL-4 on monoclonal antibody abolished the inhibitory effect of IL-4 on the bone resorption. These results suggest that IL-4 may play an important role on the inhibitory regulation of bone resorption.     

Parathyroid hormone stimulates osteoblastic expression of MCP-1 to recruit and increase the fusion of pre/osteoclasts

The clinical findings that alendronate blunted the anabolic effect of human parathyroid hormone (PTH) on bone formation suggest that active resorption is involved and enhances the anabolic effect. PTH signals via its receptor on the osteoblast membrane, and osteoclasts are impacted indirectly via the products of osteoblasts. Microarray with RNA from rats injected with human PTH or vehicle showed a strong association between the stimulation of monocyte chemoattractant protein-1 (MCP-1) and the anabolic effects of PTH. PTH rapidly and dramatically stimulated MCP-1 mRNA in the femora of rats receiving daily injections of PTH or in primary osteoblastic and UMR 106-01 cells. The stimulation of MCP-1 mRNA was dose-dependent and a primary response to PTH signaling via the cAMP-dependent protein kinase pathway in vitro. Studies with the mouse monocyte cell line RAW 264.7 and mouse bone marrow proved that osteoblastic MCP-1 can potently recruit osteoclast monocyte precursors and facilitate receptor activator of NF-kappaB ligand-induced osteoclastogenesis and, in particular, enhanced fusion. Our model suggests that PTH-induced osteoblastic expression of MCP-1 is involved in recruitment and differentiation at the stage of multinucleation of osteoclast precursors. This information provides a rationale for increased osteoclast activity in the anabolic effects of PTH in addition to receptor activator of NF-kappaB ligand stimulation to initiate greater bone remodeling.     

Structural Basis for Antibody Discrimination between Two Hormones That Recognize the Parathyroid Hormone Receptor

Parathyroid hormone-related protein (PTHrP) plays a vital role in the embryonic development of the skeleton and other tissues. When it is produced in excess by cancers it can cause hypercalcemia, and its local production by breast cancer cells has been implicated in the pathogenesis of bone metastasis formation in that disease. Antibodies have been developed that neutralize the action of PTHrP through its receptor, parathyroid hormone receptor 1, without influencing parathyroid hormone action through the same receptor. Such neutralizing antibodies against PTHrP are therapeutically effective in animal models of the humoral hypercalcemia of malignancy and of bone metastasis formation. We have determined the crystal structure of the complex between PTHrP (residues 1–108) and a neutralizing monoclonal anti-PTHrP antibody that reveals the only point of contact is an α-helical structure extending from residues 14–29. Another striking feature is that the same residues that interact with the antibody also interact with parathyroid hormone receptor 1, showing that the antibody and the receptor binding site on the hormone closely overlap. The structure explains how the antibody discriminates between the two hormones and provides information that could be used in the development of novel agonists and antagonists of their common receptor.The discovery of parathyroid hormone (PTH)6 -related protein (PTHrP) as the cause of hypercalcemia in many patients with cancer provided new insights into the pathogenesis of the skeletal complications of malignancy (1). It revealed PTHrP as a previously unrecognized hormone, related in evolution to the calcium-regulating PTH, but important in the pathogenesis of the humoral hypercalcemia of malignancy, a syndrome in which hypercalcemia occurs without evident bone metastases. Whereas PTH consists of 84 amino acids, human PTHrP has three alternative splice products of 139, 141, and 173 residues. Apart from 8 of the first 13 residues of PTH and PTHrP being identical, there is no significant identity between these peptides (2). PTHrP actively promotes bone resorption, doing so in a manner identical to that of PTH by acting upon the receptor (PTH1R) it shares with PTH. The PTH1R is located on cells of the osteoblast lineage, which program the formation and activation of osteoclasts, and on cells of the kidney tubule, through which both PTHrP and PTH promote cyclic AMP and phosphorus excretion but reduce calcium excretion. Other actions of PTHrP that reflect those of PTH include the ability to relax vascular and other smooth muscle. This response may reflect a physiological function of PTHrP rather than of PTH and is consistent with PTHrP production and local action on smooth muscles at various sites (3).The first 34 amino acids of each hormone contain the full biological activities of both PTH and of PTHrP to activate the PTH1R (4). The sequences of PTHrP and PTH between residues 14 and 34 are interesting in that, although they are not homologous, nevertheless they appear to be critical for binding of each to the seven transmembrane G protein-coupled receptor, PTH1R (4). Within the first 34 amino acids of PTH and PTHrP two functional regions have been revealed based on structural and cross-linking studies (5–8). These studies have indicated that the C-terminal half of the first 34 residues of each hormone comprises the high affinity binding domain, interacting with the N-terminal portion of the extracellular domain of the receptor. The N-terminal half of each hormone activates the receptor through contact points on the extracellular loops and juxtamembrane regions (9).Despite their equal ability to activate through the PTH1R, it was clear from the earliest work, even with antibodies against peptides within the first 14 residues of PTHrP, that highly specific antibodies could be generated that discriminate between PTH and PTHrP (10). Likewise, polyclonal antibodies against PTHrP-(1–34) that neutralized its effects completely in vitro in promotion of cyclic AMP production in response to PTHrP without any detectable neutralizing effect on PTH were used to prevent and to treat hypercalcemia in nude mice bearing xenografts of PTHrP-secreting human cancers (11, 12). Similar results were obtained with a neutralizing mouse monoclonal antibody against PTHrP (13). Subsequently, after the finding that breast cancer metastases to bone were enriched in PTHrP production (14), Guise and Mundy (15) used an experimental model in nude mice in which human breast cancer cells grow as lytic deposits in bone after intracardiac injection and showed that PTHrP production by the cancers contributed to the process of tumor establishment and growth in bone by promoting osteoclast formation and bone resorption. Furthermore, the tumor establishment and growth in bone could be prevented by treating the mice with a monoclonal antibody against PTHrP (16) or with a bisphosphonate (17) to inhibit bone resorption.The efficacy of anti-PTHrP antibodies in treating both humoral-mediated hypercalcemia in cancer and bone metastasis formation and growth in mouse models raises the prospect of humanized forms of these antibodies being used as therapeutic agents in these diseases in human subjects, and preclinical data have been obtained in support of that (18, 19). With that in mind, the present project was undertaken in which we have made use of a monoclonal antibody prepared against human PTHrP (residues 1–34), which neutralizes the actions of PTHrP through PTH1R without any action against PTH. The antibody has been complexed with recombinant human PTHrP (residues 1–108) to generate crystals that have been used to analyze the three-dimensional structure with the aim of discovering the structural basis of neutralization of PTHrP action by the antibody.     

Stimulation of extracellular signal-regulated kinases and proliferation in rat osteoblastic cells by parathyroid hormone is protein kinase C-dependent

Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10(-12) to 10(-11) m) is sufficient for substantial increases in ERK activity. Treatment with PTH-(1-34) (10(-8) m), PTH-(1-31), or 8-bromo-cAMP failed to stimulate ERKs, whereas treatment with phorbol 12-myristate 13-acetate, serum, or PTH peptides lacking the N-terminal amino acids stimulated activity. Furthermore, the activation of ERKs was prevented by pretreatment of osteoblastic cells with inhibitors of protein kinase C (GF 109203X) and MEK (PD 98059). Treatment of UMR cells with epidermal growth factor (EGF), but not PTH, promoted tyrosine phosphorylation of the EGF receptor. Transient transfection of UMR cells with p21(N17Ras) did not block activation of ERKs following treatment with low concentrations of PTH. Thus, activation of ERKs and proliferation by PTH is protein kinase C-dependent, but stimulation occurs independently of the EGF receptor and Ras activation.     

In vivo administration of 1,25-dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose-dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on the expression of the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)(2)D(3) was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 microg/kg of 1,25(OH)(2)D(3) inhibited the PTH-induced RANKL mRNA expression, but 0.5 microg/kg of the vitamin did not inhibit it. Regulator of G protein signaling-2 (RGS-2) gene expression was suppressed by 1,25(OH)(2)D(3) dose-dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)(2)D(3) suppressed PTH-induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)(2)D(3) stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH-induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor-mediated signaling.     

Parathyroid hormone-related protein (107-139) increases human osteoblastic cell survival by activation of vascular endothelial growth factor receptor-2

Parathyroid hormone-related protein (PTHrP) (107-139), in contrast to the N-terminal fragment PTHrP (1-36), has been shown to interact with the vascular endothelial growth factor (VEGF) system to modulate human osteoblast differentiation. In this study, we evaluated whether this interaction might affect human osteoblastic cell survival. Pre-incubation with PTHrP (107-139) for 1-24 h dose-dependently (0.1-100 nM) inhibited dexamethasone- or etoposide-induced cell death in human osteoblastic MG-63 cells and human osteoblast-like cells from trabecular bone. This effect, but not that elicited by PTHrP (1-36), was abolished by the VEGF receptor (VEGFR)-2 inhibitors SU5614 and SU1498 or VEGFR-2 siRNA transfection in these cells. PTHrP (107-139), but not PTHrP (1-36), at 100 nM, rapidly (within 2 min) increased VEGFR-2 tyrosine-phosphorylation in MG-63 cells; an effect unaffected by several inhibitors of metalloproteinases, neutralizing VEGF(165) or VEGFR-2 antibodies, or the VEGF binding inhibitor CBO-PP1. The latter two antagonists also failed to affect (125)I-[Tyr(116)] PTHrP (107-115) binding to these cells. Consistent with its effect on VEGFR-2 activation, PTHrP (107-139) rapidly induced extracellular signal-regulated kinase (ERK) 1/2 and Akt activaton, and both ERK and phosphatidylinsositol-3 kinase (PI3K) inhibitors abolished its pro-survival effect in human osteoblastic cells. In addition, SU5614 and the latter two types of inhibitors abrogated Runx2 activation by this peptide in MG-63 cells. Transfection with a dominant-negative Runx2 construct abolished the pro-survival effect of PTHrP (107-139), associated with a decrease in Bcl-2/Bax protein ratio. Our findings demonstrate that PTHrP (107-139) interacts with VEGFR-2 to promote human osteoblastic cell survival by a mechanism involving Runx2 activation.     

Involvement of PKC-β in PTH, TNF-α, and IL-1β Effects on IL-6 Promoter in Osteoblastic Cells and on PTH-Stimulated Bone Resorption

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-β, LY379196, was used to determine the role of the PKC-β isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) induced translocation of PKC-α and -β_I to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-β_I translocation by PTH, TNF-α or IL-1β was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-α-, or IL-1β-stimulated translocation of PKC-α. PTH, TNF-α, and IL-1β increased luciferase expression in UMR-106 cells transiently transfected with a −224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-β_I is a component of the signaling pathway that mediates PTH-, TNF-α-, and IL-1β-stimulated IL-6 expression and PTH-stimulated bone resorption.     

Parathyroid hormone and parathyroid hormone-related protein exert both pro- and anti-apoptotic effects in mesenchymal cells

During bone formation, multipotential mesenchymal cells proliferate and differentiate into osteoblasts, and subsequently many die because of apoptosis. Evidence suggests that the receptor for parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), the PTH-1 receptor (PTH-1R), plays an important role in this process. Multipotential mesenchymal cells (C3H10T1/2) transfected with normal or mutant PTH-1Rs and MC3T3-E1 osteoblastic cells were used to explore the roles of PTH, PTHrP, and the PTH-1R in cell viability relative to osteoblastic differentiation. Overexpression of wild-type PTH-1R increased cell numbers and promoted osteocalcin gene expression versus inactivated mutant receptors. Furthermore, the effects of PTH and PTHrP on apoptosis were dramatically dependent on cell status. In preconfluent C3H10T1/2 and MC3T3-E1 cells, PTH and PTHrP protected against dexamethasone-induced reduction in cell viability, which was dependent on cAMP activation. Conversely, PTH and PTHrP resulted in reduced cell viability in postconfluent cells, which was also dependent on cAMP activation. Further, the proapoptotic-like effects were associated with an inhibition of Akt phosphorylation. These data suggest that parathyroid hormones accelerate turnover of osteoblasts by promoting cell viability early and promoting cell departure from the differentiation program later in their developmental scheme. Both of these actions occur at least in part via the protein kinase A pathway.     

Involvement of PKC-beta in PTH, TNF-alpha, and IL-1 beta effects on IL-6 promoter in osteoblastic cells and on PTH-stimulated bone resorption

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-beta, LY379196, was used to determine the role of the PKC-beta isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) induced translocation of PKC-alpha and -beta(I) to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-beta(I) translocation by PTH, TNF-alpha or IL-1 beta was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-alpha-, or IL-1 beta-stimulated translocation of PKC-alpha. PTH, TNF-alpha, and IL-1 beta increased luciferase expression in UMR-106 cells transiently transfected with a -224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-beta(I) is a component of the signaling pathway that mediates PTH-, TNF-alpha-, and IL-1 beta-stimulated IL-6 expression and PTH-stimulated bone resorption.     

The activation of cAMP-dependent protein kinase is directly linked to the inhibition of osteoblast proliferation (UMR-106) by parathyroid hormone-related protein

The present study was performed to compare the effect of parathyroid hormone-related protein (PTHrP) on the proliferation of osteoblastic osteosarcoma cells (UMR-106) with that of PTH and characterize the direct involvement of cAMP in the change of osteoblast proliferation by PTHrP. Human(h)PTHrP-(1-34) (10(-11)-10(-7)M) dose-dependently inhibited [3H]thymidine incorporation (TdR) in the same manner as hPTH-(1-34). The simultaneous addition of PTHrP and PTH at a maximal effective dose of 10(-7) M did not cause additive suppressive effect on cell proliferation. Rp-cAMPs, which has been recently shown to act directly as antagonist in the activation of cAMP-dependent protein kinase (PKA), dose-dependently (10(-6)-10(-4)M) antagonized PTHrP-induced suppression of TdR in the same manner as PTH. Present study indicated that PTHrP has the same effect on osteoblast proliferation as PTH and that the activation of PKA is directly linked to the change of osteoblast proliferation by PTHrP.     

Mouse Receptor Interacting Protein 3 Does Not Contain a Caspase-Recruiting or a Death Domain but Induces Apoptosis and Activates NF-κB

The death domain-containing receptor superfamily and their respective downstream mediators control whether or not cells initiate apoptosis or activate NF-kappaB, events critical for proper immune system function. A screen for upstream activators of NF-kappaB identified a novel serine-threonine kinase capable of activating NF-kappaB and inducing apoptosis. Based upon domain organization and sequence similarity, this novel kinase, named mRIP3 (mouse receptor interacting protein 3), appears to be a new RIP family member. RIP, RIP2, and mRIP3 contain an N-terminal kinase domain that share 30 to 40% homology. In contrast to the C-terminal death domain found in RIP or the C-terminal caspase-recruiting domain found in RIP2, the C-terminal tail of mRIP3 contains neither motif and is unique. Despite this feature, overexpression of the mRIP3 C terminus is sufficient to induce apoptosis, suggesting that mRIP3 uses a novel mechanism to induce death. mRIP3 also induced NF-kappaB activity which was inhibited by overexpression of either dominant-negative NIK or dominant-negative TRAF2. In vitro kinase assays demonstrate that mRIP3 is catalytically active and has autophosphorylation site(s) in the C-terminal domain, but the mRIP3 catalytic activity is not required for mRIP3 induced apoptosis and NF-kappaB activation. Unlike RIP and RIP2, mRIP3 mRNA is expressed in a subset of adult tissues and is thus likely to be a tissue-specific regulator of apoptosis and NF-kappaB activity. While the lack of a dominant-negative mutant precludes linking mRIP3 to a known upstream regulator, characterizing the expression pattern and the in vitro functions of mRIP3 provides insight into the mechanism(s) by which cells modulate the balance between survival and death in a cell-type-specific manner.     

CARD11 and CARD14 are novel caspase recruitment domain (CARD)/membrane-associated guanylate kinase (MAGUK) family members that interact with BCL10 and activate NF-kappa B

The caspase recruitment domain (CARD) is a protein-binding module that mediates the assembly of CARD-containing proteins into apoptosis and NF-kappaB signaling complexes. We report here that CARD protein 11 (CARD11) and CARD protein 14 (CARD14) are novel CARD-containing proteins that belong to the membrane-associated guanylate kinase (MAGUK) family, a class of proteins that functions as molecular scaffolds for the assembly of multiprotein complexes at specialized regions of the plasma membrane. CARD11 and CARD14 have homologous structures consisting of an N-terminal CARD domain, a central coiled-coil domain, and a C-terminal tripartite domain comprised of a PDZ domain, an Src homology 3 domain, and a GUK domain with homology to guanylate kinase. The CARD domains of both CARD11 and CARD14 associate specifically with the CARD domain of BCL10, a signaling protein that activates NF-kappaB through the IkappaB kinase complex in response to upstream stimuli. When expressed in cells, CARD11 and CARD14 activate NF-kappaB and induce the phosphorylation of BCL10. These findings suggest that CARD11 and CARD14 are novel MAGUK family members that function as upstream activators of BCL10 and NF-kappaB signaling.     

Parathyroid hormone receptor internalization is independent of protein kinase A and phospholipase C activation

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) binding to their common receptor stimulates second messenger accumulation, receptor phosphorylation, and internalization. LLC-PK(1) cells expressing a green fluorescent protein-tagged PTH/PTHrP receptor show time- and dose-dependent receptor internalization. The internalized receptors colocalize with clathrin-coated pits. Internalization is stimulated by PTH analogs that bind to and activate the PTH/PTHrP receptor. Cell lines expressing a mutant protein kinase A regulatory subunit that is resistant to cAMP and/or a mutant receptor (DSEL mutant) that does not activate phospholipase C internalize their receptors normally. In addition, internalization of the wild-type receptor and the DSEL mutant is stimulated by the PTH analog [Gly(1),Arg(19)]hPTH-(1-28), which does not stimulate phospholipase C. Forskolin, IBMX, and the active phorbol ester, phorbol-12-myristate-13-acetate, did not promote receptor internalization or increase PTH-induced internalization. These data indicate that ligand-induced internalization of the PTH/PTHrP receptor requires both ligand binding and receptor activation but does not involve stimulation of adenylate cyclase/protein kinase A or phospholipase C/protein kinase C.     

Parathyroid hormone temporal effects on bone formation and resorption

Parathyroid hormone (PTH) paradoxically causes net bone loss (resorption) when administered in a continuous fashion, and net bone formation (deposition) when administered intermittently. Currently no pharmacological formulations are available to promote bone formation, as needed for the treatment of osteoporosis. The paradoxical behavior of PTH confuses endocrinologists, thus, a model bone resorption or deposition dependent on the timing of PTH administration would de-mystify this behavior and provide the basis for logical drug formulation. We developed a mathematical model that accounts for net bone loss with continuous PTH administration and net bone formation with intermittent PTH administration, based on the differential effects of PTH on the osteoblastic and osteoclastic populations of cells. Bone, being a major reservoir of body calcium, is under the hormonal control of PTH. The overall effect of PTH is to raise plasma levels of calcium, partly through bone resorption. Osteoclasts resorb bone and liberate calcium, but they lack receptors for PTH. The preosteoblastic precursors and preosteoblasts possess receptors for PTH, upon which the hormone induces differentiation from the precursor to preosteoblast and from the preosteoblast to the osteoblast. The osteoblasts generate IL-6; IL-6 stimulates preosteoclasts to differentiate into osteoclasts. We developed a mathematical model for the differentiation of osteoblastic and osteoclastic populations in bone, using a delay time of 1 hour for differentiation of preosteoblastic precursors into preosteoblasts and 2 hours for the differentiation of preosteoblasts into osteoblasts. The ratio of the number of osteoblasts to osteoclasts indicates the net effect of PTH on bone resorption and deposition; the timing of events producing the maximum ratio would induce net bone deposition. When PTH is pulsed with a frequency of every hour, the preosteoblastic population rises and decreases in nearly a symmetric pattern, with 3.9 peaks every 24 hours, and 4.0 peaks every 24 hours when PTH is administered every 6 hours. Thus, the preosteoblast and osteoblast frequency depends more on the nearly constant value of the PTH, rather than on the frequency of the PTH pulsations. Increasing the time delay gradually increases the mean value for the number of osteoblasts. The osteoblastic population oscillates for all intermittent administrations of PTH and even when the PTH infusion is constant. The maximum ratio of osteoblasts to osteoclasts occurs when PTH is administered in pulses of every 6 hours. The delay features in the model bear most of the responsibility for the occurrence of these oscillations, because without the delay and in the presence of constant PTH infusions, no oscillations occur. However, with a delay, under constant PTH infusions, the model generates oscillations. The osteoblast oscillations express limit cycle behavior. Phase plane analysis show simple and complex attractors. Subsequent to a disturbance in the number of osteoblasts, the osteoblasts quickly regain their oscillatory behavior and cycle back to the original attractor, typical of limit cycle behavior. Further, because the model was constructed with dissipative and nonlinear features, one would expect ensuing oscillations to show limit cycle behavior. The results from our model, increased bone deposition with intermittent PTH administration and increased bone resorption with constant PTH administration, conforms with experimental observations and with an accepted explanation for osteoporosis.