Gene expression and distribution of key bone turnover markers in the callus of estrogen-deficient, vitamin D-depleted rats.

An experimental rat model was used to test the hypothesis that in osteoporosis (OP) the molecular composition of the extracellular matrix in the fracture callus is disturbed. OP was induced at 10 weeks of age by ovariectomy and a vitamin D(3)-deficient diet, and sham-operated animals fed normal diet served as controls. Three months later a closed tibial fracture was made and stabilized with an intramedullary nail. After 3 and 6 weeks of healing, the animals were killed and the fracture calluses examined with global gene expression, in situ mRNA expression, and ultrastructural protein distribution of four bone turnover markers: osteopontin, bone sialoprotein, tartrate-resistant acid phosphatase, and cathepsin K. Global gene expression showed a relatively small number of differently regulated genes, mostly upregulated and at 3 weeks. The four chosen markers were not differently regulated, and only minor differences in the in situ mRNA expression and ultrastructural protein distribution were detected. Gene expression and composition of fracture calluses are not generally disturbed in experimental OP.

Hedgehog promotes primary osteoblast differentiation and increases PTHrP mRNA expression and iPTHrP secretion.

We used both clonal osteoblast-like cells and primary calvarial osteoblastic cells to examine the role of Hedgehog in osteoblast biology. Primary osteoblasts and several clonal osteoblast-like cell lines express Indian hedgehog (Ihh), and genes encoding both components of its receptor, patched (Ptc) and smoothened (Smo). Moreover, Ihh is relatively increased in phenotypically mature clonal cells and it increases by fivefold in primary osteoblasts as they mature in culture. Recombinant N-terminal Sonic Hedgehog (rSHH-N) upregulates Ptc and Gli-1 in osteoblasts, classical transcriptional targets. Furthermore; in response to rSHH-N, immunoreactive parathyroid hormone-related peptide (iPTHrP) secretion is transiently increased in medium conditioned by primary osteoblasts. Changes in PTHrP expression mirror those of iPTHrP, except in late cultures, when mRNA levels remain relatively elevated in response to rSHH-N. Gli-1, but not Ptc, becomes resistant to treatment with rSHH-N over a time course paralleling that of PTHrP, suggesting that mechanisms regulated by Gli-1 affect PTHrP. Last, rSHH-N increases formation of mineralized bone nodules and it accelerates expression of alkaline phosphatase, alkaline phosphatase activity, and mineralization. Taken together, these data suggest a functional role for Hedgehog protein in osteoblast recruitment and differentiation, which includes stimulation of PTHrP expression and secretion.

Sox-4 messenger RNA is expressed in the embryonic growth plate and regulated via the parathyroid hormone/parathyroid hormone-related protein receptor in osteoblast-like cells.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert potent and diverse effects in cells of the osteoblastic and chondrocytic lineages. However, downstream mediators of these effects are characterized inadequately. We identified a complementary DNA (cDNA) clone encoding the 5' end of the transcription factor Sox-4, using a subtracted cDNA library enriched in PTH-stimulated genes from the human osteoblast-like cell line OHS. The SOX-4 gene is a member of a gene family (SOX and SRY) comprising transcription factors that bind to DNA through their high mobility group (HMG)-type binding domain, and previous reports have implicated Sox proteins in various developmental processes. In situ hybridization of fetal and neonatal mouse hindlimbs showed that Sox-4 messenger RNA (mRNA) was expressed most intensely in the zone of mineralizing cartilage where chondrocytes undergo hypertrophy, and by embryonic day 17 (ED17), after the primary ossification center was formed, its expression was detected only in the region of hypertrophic chondrocytes. Sox-4 mRNA was detected in osteoblast-like cells of both human and rodent origin. In OHS cells, physiological concentrations (10(-10)-10(-9) M) of human PTH 1-84 [hPTH(1-84)] and hPT(1-34), but not hPTH(3-84), stimulated Sox-4 mRNA expression in a time-dependent manner, indicating involvement of the PTH/PTHrP receptor. Sox-4 transcripts also were detected in various nonosteoblastic human cell lines and tissues, in a pattern similar to that previously reported in mice. The presence of Sox-4 mRNA in hypertrophic chondrocytes within the mouse epiphyseal growth plate at sites that overlap or are adjacent to target cells for PTH and PTHrP, and its strong up-regulation via activated PTH/PTHrP receptors in OHS cells, makes it a promising candidate for mediating downstream effects of PTH and PTHrP in bone.

Difference in strength of autonomously replicating sequences among repeats in the rDNA region of Saccharomyces cerevisiae.

The rDNA region of Saccharomyces cerevisiae contains 100-200 tandemly repeated copies of a 9 kb unit, each with a potential replication origin. In the present studies of cloned fragments from the region involved in the regulation of replication of rDNA, we detected differences in autonomously replicating sequence (ARS) activity for clones from the same yeast strain. One clone, which showed very low ARS activity, carried a point mutation, a C instead of T, in position 9 of the essential 11 bp consensus ARS as compared to clones carrying the normal 10-of-11-bp match to the consensus. The mutation could be traced back to genomic rDNA where it represents about one-third of the rDNA units in that strain. Differences in ARS activity have implications for understanding the regulation of replication of rDNA, and the ratio of active to inactive ARS in the rDNA region may be important for potential generation of extrachromosomal copies.

Two human osteoblast-like osteosarcoma cell lines show distinct expression and differential regulation of parathyroid hormone-related protein.

Parathyroid hormone (PTH)-related protein (PTHrP) acts as a local regulator of osteoblast function via mechanisms that involve PTH/PTHrP receptors linked to protein kinase A (PKA) and C (PKC). However, the regulation of PTHrP production and mRNA expression in human osteoblasts is poorly understood. Here we have characterized alternative PTHrP mRNA 3' splicing variants, encoding PTHrP isoforms of 139, 141, and 173 amino acids, and studied the regulation of PTHrP and its mRNAs by activated PKA and PKC in two human osteoblast-like cell lines (KPDXM and TPXM). Using exon-specific Northern analysis and reverse transcriptase-coupled polymerase chain reaction, we identified mRNAs encoding PTHrP(1-139) and PTHrP(1-141) in both cell lines. PTHrP(1-139) mRNAs predominated in TPXM cells and PTHrP(1-173) mRNAs were only detected in TPXM cells. Activation of PKA or PKC resulted in different effects on PTHrP and its mRNAs in the two cell lines. In TPXM cells, peptide-specific immunoassays detected high basal levels of PTHrP, increasing by 2-fold in cell extracts and 4-fold in culture media at 7 h and 24 h after exposure to forskolin, respectively, paralleling changes in PTHrP mRNA expression. Phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), a PKC activator, had no effect. In KPDXM cells, PTHrP was not detected in culture media under basal experimental conditions, and barely detectable amounts were present in cell extracts of TPA-treated cells, although the mRNA levels increased substantially in response to TPA. In the responsive cell lines, the effects on mRNA levels were dose dependent, and increased by 6.9- to 10.5-fold and 2.0- to 4.1-fold at 4 h in TPXM and KPDXM cells after exposure to 10 microM forskolin and 150 nM TPA, respectively. PTHrP mRNA levels then declined but were sustained above controls also at 12 h in both cell lines, albeit at considerably higher levels in TPXM cells. The different responsiveness to agents activating PKA- and PKC-dependent pathways may depend on the cellular state of differentiation, or alternatively, cancer cell line-specific defects. Our data demonstrating distinct differences in mRNA species and the amounts of PTHrP produced by the two cell lines as compared with roughly equivalent overall mRNA levels may suggest that post-transcriptional mechanisms play an important role in limiting the production of intracellular and secreted PTHrPs in human osteoblastic cells.

Heterogeneity among cells that express osteoclast-associated genes in developing bone.

In the present study, we characterized the phenotype of cells in the osteoclast lineage by in situ hybridization, using antisense complementary RNA probes that encode three genes typically expressed by osteoclasts, tartrate-resistant acid phosphatase (TRAP), type IV collagenase (matrix metalloproteinase-9), and c-fms, the receptor for macrophage colony-stimulating factor. By using complementary RNA probes labeled with 35S, digoxygenin, or a combination of the two labeling methods (dual labeling in situ hybridization), we found that each of these genes exhibited a distinct expression pattern during early stages of endochondral bone development [embryonic day 15 (ED15) to ED17] in fetal mouse hind limbs. Type IV collagenase messenger RNA (mRNA) was first expressed in or just outside of the cellular layers that define perichondrium/periosteum, earlier than transcripts for TRAP or c-fms appeared at the same sites (ED15). Although transcripts for TRAP and c-fms colocalized within the skeleton, c-fms was also found in surrounding soft tissue, whereas TRAP mRNA was never detected outside the skeleton (ED16). Type IV collagenase mRNA was uniquely distributed at the chondro-osseous border, being distinct from the distribution of TRAP or c-fms (ED17). At later stages of skeletal development (ED18 to 15-day-old postnatal bone), however, there was more overlap among TRAP, type IV collagenase, and c-fms mRNAs in cells throughout bone, except at the chondro-osseous junction, where type IV collagenase continued to be uniquely localized to some cells at all developmental stages. Whereas the levels of type IV collagenase mRNA expression was most intense at the chondro-osseous margin, the levels of c-fms and TRAP mRNA expression appeared to be more uniform throughout the developing bone. The results indicate that there is considerable heterogeneity among cells expressing osteoclast-associated genes, particularly during early stages of endochondral bone development, but that this difference becomes less pronounced later in the more mature skeleton. Distinct expression patterns of these markers may represent different stages of osteoclastogenesis. Alternatively, type IV collagenase-positive and TRAP/c-fms-positive cells may represent distinct subpopulations of cells of the osteoclast lineage.

Binding and cyclic AMP stimulation by N-terminally deleted human PTHs (3-84 and 4-84) in a homologous ligand receptor system.

We have produced in yeast two human parathyroid hormone (hPTH) analogs with amino-terminal deletions, hPTH(3-84) and hPTH(4-84), employing the mating factor alpha (MF alpha) expression system. The authenticity of the polypeptides was demonstrated by amino-terminal analysis, amino acid composition, and molecular mass analysis. In cells (LLC-PK1) transfected with the human PTH/parathyroid hormone-related protein (PTHrP) receptor, using [125I-Tyr36]chickenPTHrP(1-36)NH2 as radioligand, binding studies revealed dissociation constants at equilibrium (Kd) for hPTH(3-84) and hPTH(4-84) of 4.7 and 8.0 nM, respectively, only slightly higher than natural recombinant hPTH(1-84) Kd = 2.3 nM). In comparison, [Nle8,18,Tyr34]bovinePTH(3-34)NH2 and [Tyr36]cPTHrP(1-36)NH2 showed equal Kd's of 1.9 nM. Neither of the N-terminally deleted hPTH analogs showed any detectable stimulation of cAMP production in the cells at concentrations below 20 nM. At supersaturated concentrations (500 nM) with receptor occupancy of more than 95% these hPTH analogs revealed about 15% rest agonism compared with that of hPTH(1-84). hPTH(1-84) and [Tyr36]cPTHrP(1-36)NH2 showed an equal half maximal cyclic adenosine monophosphate (cAMP) stimulation of about 0.8 and 0.7 nM, respectively. The hPTH analogs did not show any ability to antagonize cellular cAMP production induced by either hPTH or [Tyr36]cPTHrP(1-36)NH2. [Nle8,18,Tyr34]bPTH(3-34)NH2 did also not antagonize cAMP stimulation by hPTH, but inhibited [Tyr36]cPTHrP(1-36)NH2-induced cAMP production by 40% when present at a 1000 M excess. These distinct results related to PTH and PTHrP from different species are important to consider in experiments evaluating potential hPTH or PTHrP antagonism, and employment of a hPTH/PTHrP receptor model is a requirement.

Expression and characterization of a recombinant human parathyroid hormone partial agonist with antagonistic properties: Gly-hPTH(-1-->+84).

We have produced and characterized a hPTH analogue with an amino-terminal extension of glycine, Gly-hPTH(-1-->+84) (denoted Gly-hPTH). The hormone analogue was synthesized in E. coli strain BJ5183 transformed with the expression plasmid pKKPTH, extracted from the bacterial pellet and purified by reverse-phase high performance liquid chromatography. Its chemical nature, as determined by amino acid composition analysis, N-terminal amino acid analysis, and mass spectrometry, showed the 9480-Da Gly-hPTH as the predominant species. Because f-Met-Gly-hPTH was the expected form encoded by the plasmid construct, the results indicate that the f-Met residue was efficiently removed from the precurser form. The following functional characteristics of Gly-hPTH were demonstrated. 1) In cells transfected with the human PTH/PTHrP receptor, the receptor binding affinity was reduced threefold compared to the authentic hPTH(1-84) produced by Saccharomyces cerevisiae (apparent Kds: 8.4 and 2.7 nM, respectively). 2) Using the same cells, Gly-hPTH showed 27-fold reduced potency compared to hPTH(1-84) in stimulating intracellular cAMP production (EC50: 32 and 1.2 nM, respectively). 3) Gly-hPTH demonstrated antagonist activity by reducing hPTH-induced cAMP production by 33 +/- 5% (mean +/- SD) when tested at a 1:1 molar ratio. In these studies the recombinant authentic hPTH(1-84) was used as standard for comparisons, and it showed an equal receptor binding affinity and cAMP production as the chemically synthesized peptide [Nle8,18,Tyr34]bovinePTH(1-34)-NH2.

Parathyroid hormone-related protein is produced by cultured endothelial cells: a possible role in angiogenesis.

Parathyroid hormone-related protein (PTHrP) is produced by various normal and neoplastic tissues. Even if the physiological function(s) of PTHrP is unclear, evidence suggests that the protein may participate in the local regulation of smooth muscle contractility. We show here that PTHrP is produced in endothelial cells cultured from human umbilical veins as demonstrated both at the mRNA and protein level. The expression of PTHrP can be upregulated by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate, which is known to stimulate endothelial cell differentiation and angiogenesis in vitro. Unlike smooth muscle cells, the endothelial cells do not express the parathyroid hormone (PTH)/PTHrP receptor mRNA, nor could specific binding of the protein be detected. We therefore suggest that PTHrP produced by endothelial cells acts on smooth muscle cells and may be of importance for the growth and development of new vasculature.

Differences in binding affinities of human PTH(1-84) do not alter biological potency: a comparison between chemically synthesized hormone, natural and mutant forms.

The purpose of this study was to evaluate receptor binding affinities and biological properties in vitro and in vivo of various recombinant hPTH(1-84) forms representing the natural hormone and a mutagenized hPTH form, [Gln26]hPTH(1-84) (QPTH), after expression in E. coli and Saccharomyces cerevisiae. In LLC-PK1 cells stably transformed with the rat PTH/PTHrP receptor, chemically synthesized hPTH(1-84) and QPTH showed a reduced binding affinity (apparent Kd 18 and 23 nM, respectively) than the recombinant, hPTH(1-84) (apparent Kd 9.5 nM). All recombinant hPTH forms showed a similar potency to stimulate cellular cAMP production (EC50 1.5 nM) and significantly better than chemically synthesized hPTH (EC50 5.7 nM). All hormone forms showed an about equipotent activity in causing elevation in serum calcium, increased excretion of urine phosphate, and cAMP. Thus, the natural recombinant PTH forms showed higher binding affinities and adenylate cyclase activation potencies in LLC-PK1 cells, but the reduced receptor binding affinity exerted by QPTH did not transcend differences in cAMP generation and in vivo biological activities.

Synthesis of human parathyroid-hormone-related protein(1-141) in Saccharomyces cerevisiae. A correct amino-terminal processing vital for the hormone's biological activity is obtained by an ubiquitin fusion protein approach.

Gene fusions have been widely used in heterologous expression systems as a technique to stabilize the recombinant product against proteolysis, increase the translational initiation efficiency or to serve as an affinity handle for the purification of the protein. A further advantage is the potential to generate an authentic amino terminus of the foreign protein when this is vital for its biological activity, such as for the ability of human parathyroid-hormone-related protein (hPTHrP) to mediate activation of adenylate cyclase. We report here the construction and utility of a ubiquitin fusion protein system for production of the otherwise short-lived hPTHrP(1-141) as a carboxyl extension to ubiquitin in yeast. A hybrid gene containing the hPTHrP(1-141) cDNA coding region fused in-frame to the 3' end of the yeast ubiquitin cDNA was constructed and expressed under the control of the regulatable yeast metallothionein promoter. The recombinant protein was purified to homogeneity and finally characterized by N-terminal amino acid sequencing and amino acid composition analysis, demonstrating that the fusion protein was cleaved correctly and quantitatively in vivo by an ubiquitin-specific yeast endoprotease to generate authentic hPTHrP(1-141). hPTHrP(1-141) stimulated adenylate cyclase in rat osteosarcoma cell membranes to the same extent as equimolar amounts of recombinant human parathyroid hormone(1-84) and [Tyr34]hPTHrP(1-34)amide. Thus, this expression cloning strategy permits the production of authentic, biologically active recombinant hPTHrP(1-141), and the procedure can easily be adapted to make PTHrP analogues for further studies of its domain-specific activities and biological roles.

[Malignant humoral hypercalcemia and the parathyroid hormone related protein]. / Malignhumoral hyperkalsemi og det parathormonrelaterte protein.

Humoral hypercalcemia in malignant disease results from the production of humoral factors that act on bone to demineralize the skeleton, with subsequent release of calcium. It is characteristic of certain tumours without bony metastases. A recently discovered parathyroid hormone-related protein (PTHrP) has been implicated as a causative hypercalcemic agent. PTHrP exerts its calcium-mobilizing effects by interaction with parathyroid hormone (PTH) receptors in bone and kidney through its amino-terminal sequence, which is homologous with that of PTH. The human PTHrP gene could encode multiple isoforms of the protein due to alternative exon usage. Apart from its involvement in humoral hypercalcemia of malignancy, PTHrP has also been identified in normal tissues, such as keratinocytes and placenta, and is present in high concentration in milk. PTHrP may modulate the calcium homeostasis in some normal physiological conditions, probably acting in a paracrine fashion.

Regular distribution of length heterogeneities within non-transcribed spacer regions of cloned and genomic rDNA of Saccharomyces cerevisiae.

A length difference of about 50 bp in the EcoRI fragment B of the rDNA from two different strains of Saccharomyces cerevisiae has been mapped in detail by sequencing of cloned fragments. This 2.4 kb EcoRI fragment contains the start of the 35S rRNA gene at one end and the 5S rRNA gene in the middle flanked by non-transcribed spacers, NTS1 and NTS2. The difference appeared as short deletions or insertions in five regularly spaced regions within the 1 kb NTS1, 3' to the 5S rRNA gene. The same regions of heterogeneities were displayed when all available sequence data of the NTS1 were compared. Four of the variable regions are located 160-170 bp apart, indicating that they might represent linker sequences between phased nucleosomes. Two variant clones, differing in the length of one subfragment of NTS1, were isolated for each strain. In both cases these represented the major variants among chromosomal NTS1 as revealed by sequencing of genomic fragments.

Two proteolytic degradation products of calf-thymus poly(ADP-ribose) polymerase are efficient ADP-ribose acceptors. Implications for polymerase architecture and the automodification of the polymerase.

Two polypeptides with molecular masses of 76 and 59 kDa were found to copurify with poly(ADP-ribose) polymerase from calf thymus, and to be as efficient acceptors of ADP-ribose as the polymerase itself. Analysis of their CNBr fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed that the polypeptides were derived from the 112-kDa polymerase. Isolation of poly(ADP-ribose) polymerase in the absence of protease inhibitors resulted in a loss of more than 90% of the polymerase activity and an increased proportion of the 76-kDa and 59-kDa polypeptides in the final polymerase preparation. When the polymerase and the two polypeptides were separated by gel filtration or polyacrylamide gel electrophoresis in 5% acetic acid, no polymerase activity was found associated with the two fragments. Analysis of the CNBr fragments of the three polypeptides after incubation of the enzyme preparation with [32P]NAD showed that most of the fragments were radioactive, indicating multiple ADP-ribosylation sites. Several ADP-ribosylated fragments were found to be common to all three polypeptides, or to two of them.