PINP: a serum biomarker of bone formation in the rat.

Serum PINP has emerged as a reliable marker of bone turnover in humans and is routinely used to monitor bone formation. However, the effects of PTH (1-34) on bone turnover have not been evaluated following short-term treatment. We present data demonstrating that PINP is an early serum biomarker in the rat for assessing bone anabolic activity in response to treatment with PTH (1-38). Rat serum PINP levels were found to increase following as few as 6 days of treatment with PTH (1-38) and these increases paralleled expression of genes associated with bone formation, as well as, later increases in BMD. Additionally, PINP levels were unaffected by treatment with an antiresorptive bisphosphonate. PINP may be used to detect PTH-induced early bone formation in the rat and may be more generally applicable for preclinical testing of potential bone anabolic drugs.

Parathyroid hormone (hPTH 1-38) stimulates the expression of UBP41, an ubiquitin-specific protease, in bone.

Parathyroid hormone (PTH) stimulates bone formation in both animals and humans, and the expression of a number of genes has been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon, we used differential display reverse transcription polymerase chain reaction (DDRT-PCR) and screened for genes, which are differentially expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single subcutaneous (s.c.) injection of hPTH (1-38) (8 microg/100 g). We found and cloned one full-length cDNA, which encodes a putative 348 amino acid protein. Sequence analysis of this protein demonstrates a 98, 93.7, and 82.5% identity with mouse, human, and chicken ubiquitin-specific protease UBP41, respectively. Northern blot analysis confirmed that a 3.8-4 kb UBP41 mRNA transcript was rapidly increased 1 h after acute hPTH (1-38) exposure in both metaphyseal (6- to 8-fold) and diaphyseal (3-fold) bone, but returned to control levels by 24 h after exposure. In contrast, continuous exposure to hPTH (1-38), resulted in a rapid and sustained elevation of UBP41 mRNA. PTH (1-31), which stimulates intracellular cAMP, and PTHrP (1-34) both induced UBP41 mRNA expression; whereas PTH analogs (3-34) and (7-34), that do not stimulate cAMP, had no effect on UBP41 expression. UBP41 mRNA expression was also rapidly induced 1 h after injection of PGE2, but returned to the control level by 6 to 24 h. In vitro, UBP41 mRNA is expressed in primary osteoblasts (metaphyseal and diaphyseal derived) and in the osteoblast-like cell lines UMR106, ROS17/2.8, and BALC. PTH (1-38) treatment induced UPB41 expression (3.6- to 13-fold) in both primary cultures of osteoblasts and in UMR106 cells. Further analysis in UMR 106 cells demonstrated that PGE2, forskolin and dibutyryl cAMP increased UBP41 mRNA expression 4-, 4.5-, and 2.4-fold, respectively. Tissue distribution analysis of UBP41 mRNA detected transcripts in brain, heart, skeletal muscle, kidney, liver, and testis. Together, these results demonstrate that UBP41, an ubiquitin-specific protease, is selectively upregulated in bone by the osteotropic agents PTH, PTHrP, and PGE2, possibly via the PKA/cAMP pathway. We speculate that the rapid induction of UBP41 in response to these physiological regulators contributes to the mechanism by which either the structure, activity, half-life or localization of essential proteins are modified to maintain bone homeostasis.

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.

Semi-quantitative fluorescence analysis of calcein binding as a measurement of in vitro mineralization.

We describe a new, highly sensitive semiquantitative method for rapid measurement of in vitro mineralization using calcein. Fluorescence analysis of the calcein bound to the calcium phosphate (hydroxyapatite) allows direct quantitation of extracellular matrix mineral content in monolayer cultures of bone-forming cells such as primary osteoblasts or osteosarcoma cells. Osteosarcoma cell lines UMR 106 and SaOS-2 were used to demonstrate that qualitatively, calcein was bound to the same regions of the mineralized cell monolayer as seen by conventional histological staining with von Kossa or Alizarin Red S. Moreover, total bound calcein could be quantitated by direct fluorescence analysis using a Cytofluor II plate reader. Changes in cell monolayer calcein fluorescence were shown to correlate well with direct colorimetric measurement of acid-solubilized Ca+2 from parallel cultures. Relative mineral quantitation by calcein fluorescence is rapid and more sensitive than colorimetric Ca+2 assays, can be performed directly on unfixed or fixed cell monolayers, and does not require the use of radioisotopes. The cell monolayer remains intact and potentially available for further analysis.

Activation of osteocalcin transcription involves interaction of protein kinase A- and protein kinase C-dependent pathways.

Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, synthesized and secreted exclusively by osteoblastic cells in the late stage of maturation, and is considered indicator of osteoblast differentiation. Osteocalcin expression is modulated by parathyroid hormone (PTH) and a variety of other factors. The cAMP-dependent protein kinase pathway has been shown previously to have an essential role in PTH signaling and regulation of osteocalcin expression. To determine the extent to which other pathways may also participate in osteocalcin expression, we used rat and human osteoblast-like cell lines to generate stably transfected clones in which the osteocalcin promoter was fused to a luciferase reporter gene. These clones were examined for their responsiveness to agents known to activate or interfere with protein kinase A (PKA)- and protein kinase C (PKC)-dependent pathways. We have found that forskolin, cAMP, and PTH, as well as insulin-like growth factor I (IGF-I) and basic fibroblast growth factor, all were effective in activating the osteocalcin promoter. Phorbol 12-myristate 13-acetate (PMA) was also a strong inducer of the promoter, indicating that PKC plays a role in expression of osteocalcin. In combination with PTH or forskolin, the effect of PMA was additive to synergistic. Calphostin C, a selective inhibitor of PKC, decreased the PMA-, PTH-, and IGF-I-induced luciferase activity in a dose-dependent manner; a PKA inhibitor, H-89, also blocked the induction by PTH and IGF-I but not by PMA. We conclude that regulation of osteocalcin transcription is mediated by both PKA-dependent and PKC-dependent mechanisms and that the respective kinases reside on a linear or convergent pathway.

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.

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

Effect of vitamin D metabolites on the expression of alkaline phosphatase activity by epiphyseal hypertrophic chondrocytes in primary cell culture.

The effects of three vitamin D3 metabolites, 25-hydroxyvitamin D3 (25-(OH)D3), 1 alpha,25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2D3), and 24R,25-dihydroxyvitamin D3 (24R,25-(OH)2D3) on the activity of alkaline phosphatase (AP), a key enzyme involved in biomineralization, have been studied in primary cultures of chicken epiphyseal growth plate chondrocytes. Dosages of 1 alpha, 25-(OH)2D3 (10(-12) to 10(-7) M) caused a progressive, dosage- and time-dependent decrease in cellular AP levels, IC50 occurring at approximately 10(-12) M. In contrast, 24R,25-(OH)2D3 at 10(-13) to 10(-10) M stimulated cellular AP activity, half-maximal stimulation occurring at about 10(-13) M. At higher levels (10(-10) to 10(-7) M), 24R,25-(OH)2D3 caused progressive reduction in AP activity. Maximal effects of 24R,25-(OH)2D3 were evident 48 h after administration of the metabolite. 25-(OH)D3 initially (24 h) caused a weak, dosage-dependent decrease in cellular AP activity, but after 48-72 h, low levels (10(-13) to 10(-11) M) caused a dosage-dependent increase in AP activity. Higher levels of 25-(OH)D (greater than 10(-10) M) were clearly inhibitory to AP. These findings reveal that the AP activity of growth plate chondrocytes is exquisitely sensitive to both 1 alpha,25- and 24R,25-(OH)2D3 but the response to each is in opposite directions. The paradoxical response of the cells to 25-(OH)D3 can be explained if the metabolite is slowly metabolized by a 24-hydroxylase to 24R,25-(OH)2D3 leading to stimulation of cellular AP.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a new serum-free medium, USC-HC1, for growth and normal phenotype in postembryonic chicken growth plate chondrocytes.

A serum-free medium for postembryonic chicken epiphyseal growth plate chondrocytes has been developed from 104 MCDB medium. To enable these fastidious cells to survive, grow, and express normal phenotype, a substantial increase over MCDB 104 in the level of many of the amino acids was required, as well as a change in the buffer system and the addition of SerXtend, a defined, serum-free product containing various growth factors, including fibroblast growth factor. Also required was the provision of cell attachment factors, either by coating culture surfaces with type II collagen, or better, by allowing the freshly released cells to recover for several hours in a medium supplemented with 10% fetal bovine serum before plating. This new serum-free medium, which we call USC-HC1, supports growth and replication, the retention of normal polygonal morphology, the expression of significant levels of cellular alkaline phosphatase activity, the production of sulfated proteoglycans, type II collagen, and the formation of alkaline phosphatase-rich matrix vesicles by the chondrocytes. The major advantage of USC-HC1, however, is that it will provide for the first time an opportunity to examine the effects of various defined growth and hormonal factors on the phenotypic expression and differentiation of growth plate chondrocytes, in the absence of the variable (stimulatory and inhibitory) factors present in fetal bovine serum.

Isolation and characterization of calcium-accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture.

Matrix vesicles (MV) can be readily isolated from culture media of chicken growth plate hypertrophic chondrocytes grown in primary culture. The chondrocytes maintain normal morphology and synthesize type II collagen throughout the culture period. The culture-derived MV are morphologically indistinguishable from MV seen in situ and are rich in alkaline phosphatase. Formation of alkaline phosphatase-rich MV is strongly influenced by the stage of culture: large numbers are released shortly after cell seeding; marked decline is seen during cell spreading and rapid cell division; notable resurgence in alkaline phosphatase-rich MV production occurs as the cells attain confluency. Increasing the initial chondrocyte seeding density proportionately increases MV production. Cells derived from the hypertrophic region are much more capable of forming alkaline phosphatase-rich MV than those from the proliferating zone, indicating that MV formation is dependent on cellular differentiation. MV released by the cultured chondrocytes were compared in protein and phospholipid composition and in their ability to accumulate mineral ions, with plasma membrane fractions and collagenase-released MV obtained from the same tissue. Electrophoretic patterns of proteins, and the phospholipid profiles, suggest that significant modification of the plasma membrane occurs during MV formation. The vesicles are capable of accumulating large amounts of mineral ions from a metastable synthetic cartilage lymph when supplied with alkaline phosphatase substrates. This culture system thus appears to be a useful model for isolating native MV and characterizing factors required for vesicle formation and mineralization.