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J M Hock

Publications and source records attributed to J M Hock.

At least 37 records · Page 2Linked to original sources
PubMed2000

Dynamic regulation of RGS2 in bone: potential new insights into parathyroid hormone signaling mechanisms.

The initial steps involved in mediating the transduction of PTH signal via its G protein-coupled receptors are well understood and occur through the activation of cAMP and phospholipase C pathways. However, the cellular and molecular mechanisms for subsequent receptor desensitization are less well understood. Recently, a new family of GTPase activating proteins known as regulators of G protein signaling (RGS), has been implicated in desensitization of several G protein-coupled ligand-induced processes. At present, it is not known whether any of the RGS proteins play a role in PTH signaling. Using the differential display method, we screened for genes that are selectively expressed after a single s.c. injection of human PTH (1-38) (8 microg/100 g) in osteoblast-enriched femoral metaphyseal spongiosa of young male rats (3-4 weeks old). We found and cloned one full-length complementary DNA that encodes a 211-amino acid RGS protein and shares 97% sequence identity with mouse and human RGS2. Based on sequence similarity, we have designated this clone as rat RGS2. Northern blot analysis confirmed that the expression of RGS2 messenger RNA (mRNA) is rapidly and transiently increased by human PTH (1-38) in both metaphyseal (4-to 5-fold) and diaphyseal (2- to 3-fold) bone, as well as in cultured osteoblast cultures (2- to 37-fold). In vitro, forskolin and dibutyryl cAMP similarly elevated RGS2 mRNA. In vivo, PTH analog (1-31) [which stimulates intracellular cAMP accumulation, PTHrP (1-34), and prostaglandin E2] induced RGS2 mRNA expression; whereas PTH analogs (3-34) and (7-34), which do not stimulate cAMP production, had no effect on expression. In tissue distribution analysis, RGS2 is widely expressed and was detected in all tissues examined (heart, spleen, liver, skeletal muscle, kidney, and testis), with significant expression in two nonclassical PTH-sensitive tissues: the brain, and the heart. After PTH injection, RGS2 mRNA expression was induced in rat bone but not in any of the other tissues examined. These findings demonstrate that RGS2 is regulated by PTH, prostaglandin E2, and PTHrP and that regulation by PTH in bone occurs via the cAMP pathway. Additionally, these results suggest the exciting possibility that increased RGS2 expression in osteoblasts may be one of the early events influencing PTH signaling.

Amino Acid Sequence↗
PubMed2000

ADAMTS-1: A cellular disintegrin and metalloprotease with thrombospondin motifs is a target for parathyroid hormone in bone.

PTH stimulates bone formation in animals and humans, and the expressions of a number of genes have been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon we used differential display RT-PCR and screened for genes that are selectively expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single s.c. injection of human PTH-(1-38) (8 microg/100 g). We show that one of the messenger RNAs that is up-regulated in bone is ADAMTS-1, a new member of the ADAM (A disintegrin and metalloprotease) gene family containing thrombospondin type I motifs. ADAMTS-1 consists of multiple domains common to ADAM family of proteins, including pro-, metalloprotease-like, and disintegrin-like domains. However, unlike other ADAMs, ADAMTS-1 does not possess a transmembrane or cytoplasmic domain and is a secreted protein. Northern blot analysis confirmed that ADAMTS-1 was up-regulated in both metaphyseal (14- to 35-fold) and diaphyseal (4.2-fold) bone 1 h after PTH-(1-38) injection and returned to control levels by 24 h. We also analyzed the regulation of ADAMTS-1 in response to various PTH/PTH-related peptide (PTHrP) analogs and found that PTH-(1-31) and PTHrP-(1-34), which activate the protein kinase A (PKA) pathway, induce ADAMTS-1 expression 1 h after injection, whereas PTH-(3-34) and PTH-(7-34), which do not activate the PKA pathway, did not regulate expression. To investigate the effect of other osteotropic agents, we analyzed ADAMTS-1 expression after a single dose of PGE2 (6 mg/kg) and found that it was up-regulated 1 h after injection and returned to control levels by 6 h. In vitro ADAMTS-1 is expressed in primary osteoblasts and osteoblastic cell lines, but was not detectable in osteoclasts generated from macrophage colony-stimulating factor/receptor activator of NF-kappaB ligand/transforming growth factor-beta1-treated bone marrow cells. Treatment of UMR 106 osteosarcoma cells with PTH, PGE2, forskolin, or (Bu)2cAMP increased ADAMTS-1 expression 7-, 4-, 5-, and 5-fold, respectively. Also, in vitro treatment with 1alpha,25-dihydroxyvitamin D3 increased ADAMTS-1 expression 3-fold. Tissue distribution analysis showed that ADAMTS-1 is expressed at high levels in many tissues, including the heart, lung, liver, skeletal muscle, and kidney. Taken together, these results demonstrate that ADAMTS-1 is specifically up-regulated in bone and osteoblasts by the osteotropic agents PTH, PTHrP, and PGE2 possibly via the cAMP/PKA pathway. We speculate that the rapid and transient increase in ADAMTS-1 expression may contribute to some of the effects of PTH on bone turnover.

ADAM Proteins↗
PubMed2000

Six-month daily administration of parathyroid hormone and parathyroid hormone-related protein peptides to adult ovariectomized rats markedly enhances bone mass and biomechanical properties: a comparison of human parathyroid hormone 1-34, parathyroid hormone-related protein 1-36, and SDZ-parathyroid hormone 893.

Daily administration of parathyroid hormone (PTH) and PTH-related protein (PTHrP) peptides has been shown to increase bone mass and strength in animals and, for PTH, to increase bone mass in humans. Long-term direct comparison of multiple members of the PTH/PTHrP family in vivo has not been reported. We therefore selected three PTH/PTHrP molecules for direct comparison in vivo in an adult rat model of postmenopausal osteoporosis: PTH(1-34), PTHrP(1-36), and the PTH analog, SDZ-PTH 893 ¿Leu8, Asp10, Lys11, Ala16, Gln18, Thr33, Ala34 human PTH 1-34 [hPTH(1-34)]¿. A 6-month study was performed in which adult (6-month-old) vehicle-treated ovariectomized (OVX) and sham OVX rats were compared with OVX rats receiving 40 micrograms/kg per day of either PTH(1-34), PTHrP(1-36), or PTH-SDZ-893. Bone mass, as assessed by ash weight and densitometry, bone histomorphometry, biomechanical properties at trabecular and cortical sites, and indices of bone formation markedly increased in all three PTH/PTHrP peptide-treated groups as compared with controls. In general, this improvement followed a rank order of SDZ-PTH-893 > PTH > PTHrP. The adverse effect profile also was greatest with SDZ-PTH-893; these rats developed moderate hypercalcemia, marked renal calcium accumulation, and displayed a 13% mortality. These studies show that PTH(1-34), PTHrP(1-36), and PTH-SDZ-893 significantly and progressively increase bone mass and bone strength in this rat model of postmenopausal osteoporosis. The adverse effect profile correlates in general terms with efficacy. All three peptides show promise as skeletal anabolic agents. Further studies in humans will be required to define optimal efficacy-to-adverse effect ratios and relative efficacy for each peptide in human osteoporosis.

Animals↗
PubMed1999

Molecular characterization of gene expression changes in ROS 17/2.8 cells cultured in diffusion chambers in vivo.

Transplantation of diffusion chambers (DC) containing osteoblast-like cells to extraskeletal sites has been highly studied and proven to be a useful technique to investigate the process of osteoblast differentiation and bone formation. To investigate the molecular basis of osteogenesis in DC, we examined the temporal pattern of gene expression of the proliferation marker histone H4, immediate early response genes (IEGs), c-fos, c-jun, c-myc, osteoblast phenotype-associated genes, osteocalcin (OC), osteopontin (OP), type I collagen (COL1A1), alkaline phosphatase (ALP), parathyroid hormone receptor (PTHR) and matrix modifying enzyme, matrix metalloproteinase-9 (MMP-9). DC containing ROS 17/2.8 were implanted intraperitoneally into rat hosts and cultured in vivo for various times up to 56 days. Histological analysis of von Kossa stained sections of the DC contents showed a well-organized connective tissue and the production of mineralized matrices/nodules. In contrast, histological examination of DC containing Rat-2 fibroblast cells revealed the lack of an organized mineralized matrix. Molecular analysis of DC containing ROS 17/2.8 cells at 0, 3, 10, 28, and 56 days demonstrated a time-dependent decrease in DNA content associated with cell death. In the surviving cells, an increase in histone H4 mRNA (consistent with an increase in cell proliferation) was evident by 3-10 days and thereafter expression returned to control levels. In vitro, ROS 17/2.8 cells expressed detectable levels of c-fos, c-jun, c-myc, OC, OP, ALP, COL1A1, and PTHR but not MMP-9. In vivo, the expression of c-fos increased 2-fold in 3-28 days and by 56 days was 4-5 fold above control levels. In 3-10 days, c-jun expression increased 1.6-1.8-fold above control levels. In contrast, by day 28, c-jun expression decreased to control levels, but increased to 2.1-fold above control by 56 days. c-myc mRNA expression increased 3-fold within 3 days and then dropped to below control values by 10-56 days. After transplantation in vivo, the expression of OC and PTHR decreased to undetectable levels. Similarly, ALP mRNA decreased to </=28% of preimplantation values. In contrast, OPN mRNA levels increased up to 7-fold by day 10 and thereafter, returned to 1.7-fold above control values. COL1A1 mRNA decreased 2-fold at day 3 and increased to 3.5-, 1.6-, and 2.8-fold above control at days 10, 28, and 56, respectively. MMP-9 levels increased 5- to 10-fold by days 3-10, but fell to undetectable levels by 28-56 days. These results indicate that the formation of mineralized matrix (bone nodules) seen in the 56-day DC of ROS 17/2.8 cells was preceded by coordinate temporal expression of IEGs, matrix proteins, and matrix-modifying enzymes. Additionally, these results substantiate that measurement of molecular parameters in tissues formed by cells incubated in DC in vivo may be a useful predictor of the osteogenic process.

Animals↗
PubMed1999

Immunohistochemical localization of selected early response genes expressed in trabecular bone of young rats given hPTH 1-34.

Intermittent administration of parathyroid hormone (PTH) increases trabecular bone mass in vivo by stimulating bone formation. To further characterize the cellular and molecular mediators of the anabolic response to PTH, we examined the effect of intermittent synthetic hPTH 1-34 on the expression and localization of selected early response genes, c-fos, c-jun, c-myc, and IL-6 protein, in bone tissue by immunohistochemistry. Young male Sprague-Dawley rats, 70-100 g, were injected s.c. with 8 microg/100 g PTH or vehicle control, once daily for 5 days. Femurs were harvested 1 and 24 hours after the fifth injection, then fixed, decalcified, processed for wax embedding, and sections were immunostained. Early response genes, c-fos, c-jun and IL-6, were strongly expressed in osteoblasts, osteocytes, and megakaryocytes in bones 1 hour after PTH, when compared with vehicle-treated controls or sections from rats, 24 hours after PTH injection. Osteoblasts, osteocytes, and megakaryocytes were also positive for c-myc but the differences in stain intensity between control and treated groups were marginal. Also, scattered islands of hematopoietic cells in the marrow stained intensely for IL-6 by 1 hour after PTH, but the stain intensity decreased to control level 24 hours after the last PTH injection. Scattered islands of hematopoietic cells in the bone marrow stained more strongly for c-fos than either c-jun or c-myc, but neither localization nor stain intensity were regulated by PTH at the time points examined. We conclude that during the immediate early phase of the anabolic response, PTH regulates c-fos, c-jun, and IL-6 expression in osteoblasts, osteocytes, megakaryocytes, and selected bone marrow hematopoietic cells in bone.

Administration, Cutaneous↗
PubMed1999

Daily treatment with human recombinant parathyroid hormone-(1-34), LY333334, for 1 year increases bone mass in ovariectomized monkeys.

PTH stimulates bone formation to increase bone mass and strength in rats and humans. The aim of this study was to determine the skeletal effects of recombinant human PTH-(1-34) [rhPTH-(1-34)] in monkeys, as monkey bone remodeling and structure are similar to those in human bone. Adult female cynomolgus monkeys were divided into sham-vehicle (n = 21), ovariectomized (OVX)-vehicle (n = 20), and OVX groups given daily s.c. injections of rhPTH-(1-34) at 1 (n = 39) or 5 (n = 41) microg/kg for 12 months. Whole body bone mineral content was measured, as was bone mineral density (BMD) in the spine, proximal tibia, midshaft radius, and distal radius. Serum and urine samples were also analyzed. rhPTH-(1-34) treatment did not influence serum ionized Ca levels or urinary Ca excretion, but depressed endogenous PTH while increasing serum calcitriol levels. Compared to that in the OVX group, the higher dose of rhPTH-(1-34) increased spine BMD by 14.3%, whole body bone mineral content by 8.6%, and proximal tibia BMD by 10.8%. Subregion analyses suggested that the anabolic effect of rhPTH-(1-34) on the proximal tibia was primarily in cancellous bone. Similar, but less dramatic, effects on BMD were observed with the lower dose of rhPTH-(1-34). Daily s.c. rhPTH-(1-34) treatment for 1 yr increases BMD in ovariectomized monkeys without inducing sustained hypercalcemia or hypercalciuria.

Animals↗
PubMed1999

Comparison of recombinant human PTH(1-34) (LY333334) with a C-terminally substituted analog of human PTH-related protein(1-34) (RS-66271): In vitro activity and in vivo pharmacological effects in rats.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are believed to exert their biological actions through binding and activation of a common cell surface receptor. Recently, an analog of PTHrP (RS-66271), was described that demonstrated reduced binding affinity for the PTH/PTHrP receptor compared with bovine PTH(1-34) but retained equal biological activity. The present study investigated the receptor binding affinities of synthetic RS-66271 and recombinant human PTH(1-34) (LY333334) and compared their in vitro and in vivo pharmacological effects. RS-66271 had one hundredth the activity of PTH(1-34) in competing for the binding of [125I] [Nle8,18, Tyr34]human PTH(1-34) to the human PTH/PTHrP receptor stably expressed in a human kidney cell line. Despite this reduced binding affinity, RS-66271 had equivalent activity in increasing both cAMP production in osteoblast-like cells and bone resorption in neonatal mouse calvariae. However, RS-66271 was 7. 6-fold less active in stimulating inositol phosphate production. For in vivo studies, young, male Fisher rats received a daily subcutaneous dose of either 10 or 40 microg/kg of peptide for 1, 2, or 4 weeks. Volumetric bone mineral density and total bone mineral content of the proximal tibia were determined by peripheral quantitative computerized tomography. Trabecular and cortical bone of the distal femur were analyzed for calcium and dry weight. Lumbar vertebrae (L4-L6) were analyzed by histomorphometry. Trabecular and cortical bone mass showed a dose- and time-dependent increase in the treated animals compared with the controls. These increases were evident as early as 1 week after initiation of dosing. There were no consistent significant differences in the comparative effects of PTH(1-34) and RS-66271 on the measured bone parameters. In conclusion, despite the reduced binding affinity of RS-66271 for the PTH/PTHrP receptor compared with human PTH(1-34), both peptides displayed similar in vitro and in vivo pharmacological effects.

Animals↗
PubMed1999

Anabolic effects of human biosynthetic parathyroid hormone fragment (1-34), LY333334, on remodeling and mechanical properties of cortical bone in rabbits.

Intermittent administration of parathyroid hormone (PTH) has an anabolic effect in cancellous bone of osteoporotic humans. However, the effect of PTH on cortical bone with Haversian remodeling remains controversial. The aim of this study was to determine the effects of biosynthetic human PTH(1-34) on the histology and mechanical properties of cortical bone in rabbits, which exhibit Haversian remodeling. Mature New Zealand white rabbits were treated with once daily injections of vehicle, or PTH(1-34), LY333334, at 10 micrograms/kg/day or 40 micrograms/kg/day for 140 days. Body weight in rabbits treated with PTH did not change significantly over the experimental period. Serum calcium and phosphate were within the normal range, but a 1 mg/ml increase in serum calcium was observed in rabbits given the higher dose of PTH. Histomorphometry of cortical bone in the midshaft of the tibia showed significant increases in periosteal and endocortical bone formation in these rabbits. Intracortical bone remodeling in the tibia was activated and cortical porosity increased by PTH. Cross-sectional bone area and bone mass of the midshaft of the femur increased significantly after PTH treatment. Ultimate force, stiffness, and work to failure of the midshaft of the femur of rabbits given the 40 micrograms dose of PTH were significantly greater than those in the control group, whereas elastic modulus was significantly lower than that in the rabbits given the 10 micrograms dose of PTH, but not different from controls. In the third lumbar vertebra, PTH increased both formation and resorption without increasing cancellous bone volume. The increases in bone turnover and cortical porosity were accompanied by concurrent increases in bone at the periosteal and endocortical surfaces. The combination of these phenomena resulted in an enhancement of the ultimate stress, stiffness, and work to failure of the femur.

Animals↗
PubMed1998

Intermittent administration of parathyroid hormone (1-34) stimulates matrix metalloproteinase-9 (MMP-9) expression in rat long bone.

Intermittent doses of parathyroid hormone (PTH) stimulate bone formation in animals and humans, but the molecular mechanisms underlying this phenomenon are not understood. Bone formation culminates with the expression of type I collagen, osteocalcin, and alkaline phosphatase, but genes that initiate and support the anabolic response are not known. To identify novel PTH-regulated genes in bone during the anabolic response, we used differential display-polymerase chain reaction (DDRT-PCR) to analyze RNA from young male rats injected with either human PTH (1-34) or vehicle control, once daily for 5 days. Total RNA was isolated from the distal femur metaphysis at 1, 6, and 48 h after the final injection and subjected to DDRT-PCR. We identified three PTH-responsive transcripts as matrix metalloproteinase-9 (MMP-9), creatine kinase, and the alpha1 (I) polypeptide chain (COL1A1) of type I collagen. The concomitant upregulation of MMP-9 and COL1A1 during bone formation was particularly intriguing. Further characterization of MMP-9 expression revealed that it was localized to osteoblasts, osteocytes, megakaryocytes, and cells of the bone marrow in the rat distal femur metaphysis. Northern analysis for MMP-9 expression in other tissues indicated that this transcript was present in the kidney and brain. In vitro, PTH regulated the protein synthesis of MMP-9 by osteoblasts of the primary spongiosa. We propose that PTH may promote bone formation by mediating the subtle variation in MMP activities, thus preparing the extracellular matrix for the subsequent bone cell migration and deposition of new osteoid.

Amino Acid Sequence↗
PubMed1998

Recruitment and proliferative responses of osteoblasts after mechanical loading in vivo determined using sustained-release bromodeoxyuridine.

Mechanical bending of a rat's tibia in vivo can increase endocortical bone formation by over sixfold. It has been proposed that mechanical loading increases bone formation by driving osteoprogenitor cells in the marrow stroma to progress through the cell cycle and subsequently differentiate into osteoblasts at the cortical bone surfaces. We used a sustained-release preparation of 5-bromo-2'-deoxyuridine (SR-BrdUrd) to determine the origin of endocortical osteoblasts in rat tibiae after mechanical loading. SR-BrdUrd was bioavailable for the entire 96 h duration of the experiments, so all cells that progressed through a cell cycle were labeled with BrdUrd. Although the endocortical osteoblast surface was significantly increased (p < 0.05) at 48 h after loading, the percentage of BrdUrd-labeled osteoblasts did not increase, suggesting that the newly differentiated osteoblasts on the endocortical surface did not originate from proliferating cells. At 96 h after loading, 30-40% of the endocortical osteoblasts were BrdUrd labeled. The majority of BrdUrd-labeled osteoblasts appeared on the endocortical bone surface within the third day after loading, indicating that proliferation and differentiation of precursors into endocortical osteoblasts required 72 h after the loading stimulus. These results indicate that mechanical loading can cause two distinct osteoblastic responses: an immediate response within 48 h in which osteoblasts are recruited from nondividing preosteoblasts and/or bone-lining cells, and a delayed response involving proliferation and differentiation of preosteoblasts that requires > or =3 days.

Animals↗
PubMed1998

Osteoprogenitor cells as targets for ex vivo gene transfer.

We transduced osteoprogenitor cells with recombinant retrovirus and analyzed proviral integration patterns into chromosomal DNA to detect for the first time the clonal and cellular fate of osteoprogenitor-derived progeny cells. Metaphyseal bone cells and diaphyseal stromal cells were isolated from the distal femurs of young rats, transduced with the vM5neolacZ recombinant retrovirus, and selected in the neomycin analog, G418. Following surgical marrow ablation of a femur in one leg of mature rats, retroviral-transduced metaphyseal or diaphyseal cells were injected into the ablated site. These rats were killed 5-6 days later. Metaphyseal and diaphyseal cells were isolated from distal femurs, selected in G418, and stained for beta-galactosidase (beta-gal+). The number and clonal origin of transduced progenitor cells were determined. High numbers of beta-galactosidase colonies with an osteoblast phenotype were obtained following metaphyseal transplants and detected in 100% of metaphyseal and none of diaphyseal specimens. In contrast, beta-galactosidase colonies derived from diaphyseal transplants were detected in 50% of specimens in both the metaphysis and diaphysis, and the absolute number of progenitor cell colonies was 60-fold less than metaphyseal transplants. Provirus was only detected in the ablated bones and not in the contralateral bone or other tissues. Proviral integration fragment analysis showed a single integration site for recovered metaphyseal cell clones, consistent with their origination from a common single progenitor. This is one of the first demonstrations of successful transplantation of clonal osteoprogenitors to their site of origin in bone. It may be possible to use these cells to target genes to bone for therapeutic use in skeletal and hematopoietic diseases.

Animals↗
PubMed1997

Topoisomerase II expression in osseous tissue.

The molecular mechanisms that mediate the transition from an osteoprogenitor cell to a differentiated osteoblast are unknown. We propose that topoisomerase II (topo II) enzymes, nuclear proteins that mediate DNA topology, contribute to coordinating the loss of osteoprogenitor proliferative capacity with the onset of differentiation. The isoforms topo II-alpha and -beta, are differentially expressed in nonosseous tissues. Topo II-alpha expression is cell cycle-dependent and upregulated during mitogenesis. Topo II-beta is expressed throughout the cell cycle and upregulated when cells have plateaued in growth. To determine whether topo II-alpha and -beta are expressed in normal bone, we analyzed rat lumbar vertebrae using immunohistochemical staining. In the tissue sections, topo II-alpha was expressed in the marrow cavity of the primary spongiosa. Mature osteoblasts along the trabecular surfaces did not express topo II-alpha, but were immunopositive for topo II-beta, as were cells of the marrow cavity. Confocal laser scanning microscopy was used to determine the nuclear distribution of topo II in rat osteoblasts isolated from the metaphyseal distal femur and the rat osteosarcoma cells, ROS 17/2.8. Topo II-alpha exhibited a punctate nuclear distribution in the bone cells. Topo II-beta was dispersed throughout the interior of the nucleus but concentrated at the nuclear envelope. Serum starvation of the cells attenuated topo II-alpha expression but did not modulate expression of the beta-isoform. These results indicate that the loss of osteogenic proliferation correlates with the downregulation of topo II-alpha expression.

Animals↗
PubMed1997

Parathyroid hormone (1-34)-mediated interleukin-6 induction.

Parathyroid hormone (PTH) functions in part by regulating osteoblast cytokine expression. We recently demonstrated that PTH induced a rapid and transient increase in interleukin-6 (IL-6) mRNA expression in rat bones in vivo. To determine the molecular basis of this effect, we analyzed the human IL-6 promoter fused (-1,179 to +9) with the chloramphenicol acetyltransferase (CAT) reporter gene in stable transfections into human osteoblast-like osteosarcoma SaOS-2 cells. We compared the effects of PTH on IL-6 expression with adenylate cyclase activator forskolin, PKC activator phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, interleukin-1 alpha (IL-1 alpha), prostaglandin E-2 (PGE-2), RS-66271 (a parathyroid hormone-related peptide analog), and platelet-derived growth factor-BB (PDGF-BB). Analyses of cell clones showed that IL-6 promoter expression was extremely low in the unstimulated state. Exposure to PTH (0.001-100 nM) for 12 h stimulated CAT expression in a dose-dependent manner (200-500% of control). Treatment with IL-1 alpha was more potent than PTH in inducing transcription of the IL-6 promoter (900-1,000%). Activation of the cAMP-PKA pathway by treatment with forskolin induced a comparable level of induction with PTH. Together, the effects of PTH and forskolin were additive. RS-66271, previously shown to have PTH-like effects, induced a comparable level of IL-6 promoter expression. When examined together, PTH+RS-66271 effects were comparable to PTH effects alone. Exposure to PGE-2, PMA, PDGF-BB, or A23187 for 12 h did not significantly alter IL-6 promoter expression. These results demonstrate PTH, forskolin, the PTHrP analog RS-66271, and IL-1 alpha stimulate IL-6 expression by stimulating gene transcription. The response to forskolin suggests that the messenger system mediated by PKA is sufficient to induce IL-6 expression.

Becaplermin↗
PubMed1997

Proliferating cells in the primary spongiosa express osteoblastic phenotype in vitro.

We have shown that intermittent parathyroid hormone (PTH) treatment targets proliferating cells in the primary spongiosa of trabecular bone of young rats, resulting in an increased number of osteoblasts. To further characterize these proliferating osteoprogenitor cells, bromodeoxyuridine (BrdUrd) incorporated in vivo, was used as a marker to identify and isolate cells for in vitro studies. Proliferating cells were labeled in vivo in young rats with BrdUrd and 24 h later were isolated by trypsinization of sections of the primary spongiosa of the distal femur metaphysis. Within 12 h of isolation, BrdUrd+ cells formed distinct foci containing 20-500 cells with fibroblast morphology. Stimulation of proliferation as determined by [3H]-thymidine incorporation was observed for these cells in response to fetal bovine serum, platelet derived growth factor, and transforming growth factor beta-1. Neither insulin-like growth factor-1 (IGF-1) nor insulin stimulated proliferation PTH (1-34) and dexamethasone inhibited proliferation. The effects of PTH and dexamethasone were additive. Cells expressed the osteoblast phenotype as evidenced by synthesis of type I collagen, expression of high alkaline phosphatase activity, and production of increased intracellular cAMP in response to PTH (1-34). Confluent cell aggregates spontaneously formed mineralized nodules within 4-7 days, in the absence of inducers. These observations suggest that the primary spongiosa cells recapitulates the differentiation process in vitro in an accelerated fashion and may serve as a useful model to study osteoblast differentiation.

Animals↗
PubMed1996

A role for CD8+ T lymphocytes in osteoclast differentiation in vitro.

Bone loss associated with chronic inflammatory diseases has been attributed to the release of cytokines from T lymphocytes. However, the role of T lymphocyte subsets in the mediation of osteoclast activity has not been extensively studied. Cocultures of murine bone marrow and BALC cells (murine calvarial-derived cell line) were used to study osteoclast differentiation. Murine marrow was left intact or depleted of cells expressing the CD8 or CD4 antigen by immunomagnetic separation and then cocultured with BALC cells in the presence or absence of 1,25-(OH)2D3. Depleting bone marrow of CD4-positive (CD4+) cells did not affect osteoclast differentiation (formation of tartrate-resistant acid phosphatase positive cells with three or more nuclei). However, depletion of CD8-positive (CD8+) cells resulted in a 40% increase in the number of osteoclasts formed. Addition of CD8+ cells to CD8+ cell depleted cocultures via Transwells abolished the stimulatory effects on osteoclast differentiation resulting from CD8+ cell depletion. Neutralizing antibodies to interleukin-4 and transforming growth factor-beta did not affect osteoclast differentiation in these cultures. These findings suggest that CD8+ cells may be involved in the regulation of osteoclast differentiation and that this effect is not mediated by interleukin-4 or transforming growth factor-beta.

Animals↗
PubMed1995

Growth hormone does not enhance the anabolic effect of human parathyroid hormone (1-34) on bone in aging multiparous and virgin rats.

In humans, the progressive loss in skeletal bone mass with aging increases the risk of osteoporosis and bone fractures in the elderly. As parathyroid hormone (PTH) and growth hormone (GH) may both be anabolic in bone, we tested if the combination of these hormones would increase bone mass more than either agent alone in aging multiparous and virgin female rats. In four separate studies, female rats (15-18 months old) were treated for 12 or 15 days with synthetic human parathyroid hormone (hPTH 1-34) at 80 micrograms/kg per day given once daily, alone or combined with ovine GH at 1 mg/kg per day given twice daily. Bone of distal femurs, proximal tibias and lumbar vertebrae, dietary mineral (Ca, P, Mg) excretion and balance and serum chemistry were assessed. PTH alone stimulated bone formation and increased bone mass in three of four experiments and consistently increased serum 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and mineral retention. GH alone did not change either bone formation or bone mass, but combined with PTH increased mineral retention and serum 1,25-(OH)2D3), more than either hormone alone. Despite this stimulatory effect on Ca retention, GH did not further enhance the anabolic effect of hPTH 1-34 on bone mass of aging multiparous and virgin female rats.

Aging↗
PubMed1995

Anabolic effect of parathyroid hormone is not modified by supplementation with insulinlike growth factor I (IGF-I) or growth hormone in aged female rats fed an energy-restricted or ad libitum diet.

PTH increases bone mass by stimulating bone formation in vivo, and IGF-I partially mediates the stimulatory effect of PTH on collagen synthesis in vitro. The objectives of this study were to determine if the anabolic effect of PTH in aged rats was diminished by an energy-restricted (ER) diet in which IGF-I levels have been shown to be decreased, and if GH or IGF-I modified the bone response to PTH. Two strains of aged female rats, 18-24 months, fed either ad libitum (AL) or 60% ER diets since weaning, were treated subcutaneously (SC) with 8 micrograms/100 g body weight hPTH 1-34 alone or in combination with GH given SC as 0.1 mg/100 g body weight twice daily for 24 or 32 days. Additional ER-fed rats were supplemented SC with 60 micrograms/100 g body weight rhIGF-I twice daily and treated with PTH and/or GH for 12 days. Histomorphometric indices of bone volume (BV/TV), mineral apposition rate (MAR) and bone formation rate (BFR/BS) in lumbar vertebrae and bone mineral content (BMC) of tibia increased in all PTH-treated rats. BV significantly increased after 32 (p < 0.01) or 24 (p < 0.05) days, but not 12 days (NS). Significant increases in mineralizing surfaces (MS/BS) and MAR resulted in 2.9-3.6-fold increases in BFR associated with PTH treatment alone or with GH (p < 0.01). These increases occurred regardless of an ER diet, and were not modified by IGF-I or GH. Although ER rats weighed less, femur length was not affected and serum chemistry values were within the physiologic range for rats. In summary, PTH increased bone mass by stimulating bone formation in aged rats irrespective of diet, and supplementation with GH or IGF-I did not modify the response of bone to PTH.

Aging↗