Parathyroid Hormone-related Peptide mRNA Expression Research Articles

Gene and protein expression of cartilage canal and osteochondral junction chondrocytes and full-thickness cartilage in early equine osteochondrosis

The objective of this study was to investigate the expression of several regulatory factors associated with cartilage maturation in horses with early osteochondrosis (OC) compared to normal controls. The hypothesis was that expression levels of Indian hedgehog (Ihh), parathyroid hormone-related peptide (PTH-rP), vascular endothelial growth factor (VEGF), platelet-derived growth factor-A (PDGF-A), and matrix metalloproteinase-13 and -3 (MMP-13, -3) would be increased in OC. Articular cartilage and osteochondral samples were collected from the femoropatellar joints from seven OC and eight normal young (1-6 months) horses after euthanasia and snap frozen or suspended in 4% paraformaldehyde. Laser capture microdissection was used to capture cells surrounding cartilage canals and the osteochondral junction. Total RNA was isolated from whole cartilage and laser-captured cells. Equine-specific Ihh, PTH-rP, VEGF, PDGF-A, MMP-13, and MMP-3 mRNA expression levels were evaluated by real-time (RT)-PCR. Spatial tissue protein expression was determined by immunohistochemistry. In laser-captured samples, there was significantly increased MMP-13 and PDGF-A gene expression in chondrocytes adjacent to cartilage canals and increased PDGF-A gene expression in osteochondral junction chondrocytes of OC-affected foals. In full-thickness cartilage samples, there was significantly increased Ihh, MMP-3, and MMP-13 gene expression in OC samples, while PTH-rP protein expression was significantly higher along the osteochondral junction. The results suggest that pathways involving cartilage maturation and ossification are altered in early OC and may be associated with disease pathogenesis.

Extracellular calcium regulates parathyroid hormone-related peptide expression in osteoblasts and osteoblast progenitor cells

Parathyroid hormone-related peptide (PTHrP) has been shown to have anabolic effects on bone in women with postmenopausal osteoporosis. On the cellular level PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The calcium concentration is considerably higher in the vicinity of resorbing osteoclasts than in the plasma. Therefore the osteoblasts are likely to be confronted by elevated extracellular calcium concentrations in the areas of resorptive activity. The present study was designed to assess the possibility that extracellular calcium could regulate PTHrP expression in osteoblastic cells. Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The PTHrP release into the culture media was measured by an immunoradiometric assay and the expression of PTHrP, osteocalcin and Runx2 mRNA was assayed by real-time PCR. Increasing the extracellular calcium from 1 mM to 5 mM for 24 h resulted in a 4–6-fold increase in the PTHrP release. PTHrP mRNA was also increased by elevated calcium levels. The effect of calcium stimulation on PTHrP release could be seen within 60 min of treatment. The extracellular calcium sensing receptor (CaR) agonist neomycin mimicked the effects of calcium and the MEK/MAPK inhibitor PD98059 abolished the effect of calcium and neomycin. High extracellular calcium increased the mineralization of hMSC and the expression of osteocalcin, but this effect was not mimicked by neomycin. Our results show that in hMSC, elevated extracellular calcium levels increases both released PTHrP and PTHrP mRNA expression. The effect of calcium on PTHrP can be mimicked by activation of the CaR and can be diminished by inhibition of the MAPK signalling pathway.

Induction of parathyroid hormone‐related peptide following peripheral nerve injury: Role as a modulator of Schwann cell phenotype

Parathyroid hormone-related peptide (PTHrP) is widely distributed in the rat nervous system, including the peripheral nervous system, where its function is unknown. PTHrP mRNA expression has recently been shown to be significantly elevated following axotomy of sympathetic ganglia, although the role of PTHrP was not investigated. The role of PTHrP in peripheral nerve injury was investigated in this study using the sciatic nerve injury model and dorsal root ganglion (DRG) explant model of nerve regeneration. We find that PTHrP is a constitutively secreted peptide of proliferating Schwann cells and that the PTHrP receptor (PTH1R) mRNA is expressed in isolated DRG and in sciatic nerve. Using the sciatic nerve injury model, we show that PTHrP is significantly upregulated in DRG and in sciatic nerve. In addition, in situ hybridization revealed significant localization of PTHrP mRNA to Schwann cells in the injured sciatic nerve. We also find that PTHrP causes a dramatic increase in the number of Schwann cells that align with and bundle regrowing axons in explants, characteristic of immature, dedifferentiated Schwann cells. In addition to stimulating migration of Schwann cells along the axonal membrane, PTHrP also stimulates migration on a type 1 collagen matrix. Furthermore, treatment of purified Schwann cell cultures with PTHrP results in the rapid phosphorylation of the cAMP response element protein, CREB. We propose that PTHrP acts by promoting the dedifferentiation of Schwann cells, a critical requirement for successful nerve regeneration and an effect consistent with known PTHrP functions in other cellular differentiation programs.

Parathyroid hormone-related peptide expression in rat collagen-induced arthritis

The aim of this study was to describe expression of parathyroid hormone-related peptide (PTHrP) in collagen-induced arthritis (CIA), a well-established animal model for rheumatoid arthritis. CIA was induced in female dark agouti rats. Inguinal (ILNs) and popliteal (PLNs) lymph nodes and distal interphalangeal joints (DIP) were retrieved at different time points. Tissues were processed for detection of PTHrP and cell marker proteins by immunohistochemistry. Lymph node RNA was extracted, and PTHrP mRNA quantified using competitive reverse transcriptase polymerase chain reaction. Hyperplasia of ILNs was observed 2 days after injection, coinciding with the peak in PTHrP expression in ILNs (1240 +/- 373 gene copies/ng RNA vs normal 339 +/- 120, P < 0.05). Hyperplasia of PLNs was first seen at 1 day after onset of arthritis, coinciding with the peak in PTHrP expression in PLNs (2267 +/- 697 vs normal 781 +/- 136, P < 0.01). PTHrP expression in PLNs remained increased 5 days after onset (1361 +/- 302 vs normal 781 +/- 136, P < 0.05). In both PLNs and ILNs PTHrP protein was localized to high endothelial venules, lymphocytes and monocytes/macrophages. In DIP joint synovium PTHrP staining was first detected on day 10 after onset, and was most abundant at day 20 after onset, at sites of bone resorption and deposition, where it was localized to neutrophils, cells of monocyte lineage and osteoblasts. Changes in ILN and PLN PTHrP mRNA expression suggest that elevated levels of the cytokine are associated with aggravation of the inflammatory immune response. Changes in PTHrP in DIP joints indicate its involvement in late rather than early pathogenic events in CIA joints.

Regulation of chondrogenesis by PTH/PTHrP signaling

During endo chondral bone development, parathyroid hormone-related peptide(PTHrP)is expressed by the perichondrial cells and proliferating chondrocytes in the periarticular region. PTHrP mRNA expression is increased by Indian Hedgehog(Ihh), which is expressed by prehypertrophic and hypertrophic chondrocytes. PTHrP acts directly on columnar proliferating chondrocytes to prevent hypertrophic differentiation. This prevention indirectly results in less Ihh signaling by decreasing the number of prehypertrophic and hypertrophic chondrocytes expressing Ihh. This negative feedback loop serves to regulate the balance of differentiation and proliferation of growth plate chondrocytes. Gs, cAMP, Sox9 and CREB seem to act downstream of PTHrP, while Patched, smoothened, Gli appear to act downstream of Ihh.

Preventive effects of parathyroid hormone-related peptide on stress-induced gastric hypercontraction in the rat.

Parathyroid hormone-related peptide (PTHrP) appears to be a potent smooth muscle relaxant, but there has been no study of its effects on gastric motility in vivo. The purposes of this study were to evaluate the effects of external PTHrP on stress-induced gastric motility in vivo and on the expression of PTHrP and PTH/PTHrP receptors in the rat stomach. Stress-induced hypercontraction was evoked by restraint water immersion (RWI). Gastric motility was evaluated with a strain gauge force transducer, and the effects of external PTHrP-(1-34) (10 micro i.p.) on gastric motility were examined. Expressions of PTHrP and PTH/PTHrP receptor mRNA were evaluated by RNase protection assay. External PTHrP significantly suppressed abnormal contraction and mucosal lesions upon RWI stress. Upon RWI stress, the expression of PTHrP mRNA decreased, but that of PTH/PTHrP receptor mRNA was enhanced reciprocally. The PTH/PTHrP receptor was localized in smooth muscle cells of the muscle layers immunohistochemically. These findings suggest that smooth muscle contractile activity is modified by the autocrine/paracrine mechanism of PTHrP in the rat stomach and that the external PTHrP prevents stress-induced hypercontraction and mucosal lesions.

Parathyroid hormone-related peptide and Indian hedgehog expression patterns in naturally acquired equine osteochondrosis

Early changes in parathyroid hormone-related peptide (PTH-rP) and Indian hedgehog (Ihh) expression were examined in equine articular osteochondrosis (OC) as a model of a naturally acquired dyschondroplasia. Cartilage was harvested from OC-affected femoropatellar or scapulohumeral joints from immature horses and normal control horses of similar age. PTH-rP expression levels were assessed by semi-quantitative PCR, in situ hybridization, and immunohistochemistry. Ihh protein expression levels were assessed by immunohistochemistry. Elevated PTH-rP protein and mRNA expression were identified in the deeper layers of affected articular cartilage and the fibrous tissue of interposing clefts. These changes were confined to the chondrocytes in the OC-affected cartilage, which had significantly increased PTH-rP protein and mRNA expression when compared to control cartilages. Ihh protein expression showed similar distribution as PTH-rP in the deeper layers of articular cartilage; however, only a trend for increased Ihh immunostaining was evident in the OC cartilage when compared to the normal cartilage. Increased PTH-rP expression in pre-hypertrophic chondrocytes of diseased OC cartilage suggests a possible link between this peptide and the delayed ossification, which is a consistent histologic alteration in OC. More evidence is necessary to determine the role of Ihh in articular cartilage and if a similar feedback cycle exists as previously described for the growth plate.

Prohormone convertase furin has a role in gastric cancer cell proliferation with parathyroid hormone-related peptide in a reciprocal manner.

We investigated the expression of parathyroid hormone-related peptide (PTHrP) and the relationship between PTHrP and its endoprotease furin in gastric cancer. PTHrP was colocalized with furin in 75% of gastric cancer tissues (six of eight) from patients with high serum PTHrP levels. PTHrP mRNA expression was confirmed in 67% of gastric cancer cell lines (four of six), whereas furin mRNA was detected in all six gastric cancer cell lines. In a cultured gastric cancer cell line, MKN28, mature PTHrP protein expression was markedly increased by transfection of furin cDNA. Furin cDNA-transfected MKN28 cells grew faster than did the mock controls. Moreover, furin mRNA expression in cultured gastric cancer cells was enhanced when PTHrP was added to the culture medium. These results suggest a link between PTHrP and furin in the regulation of gastric cancer cell growth. Furin might be involved not only in the production of the mature form of PTHrP, but also in promoting growth in gastric cancer cells.

TGF- β1Downregulates PTHrP in Coronary Endothelial Cells

Parathyroid hormone-related peptide (PTHrP) is expressed throughout the cardiovascular system including coronary endothelial cells. Factors involved in the regulation of cardiac PTHrP expression have not been examined before. This study investigates the influence of transforming growth factor (TGF)- β1on ventricular PTHrP expression. Coronary endothelial cells were isolated from ventricles of adult rats and PTHrP protein expression in these cultures was analysed by immunoblotting. TGF- β1caused a concentration-dependent reduction in PTHrP protein within 24 h. In transgenic mice over-expressing TGF- β1ventricular PTHrP protein expression and release was reduced compared to non-transgenic littermates. Similar concerns hold for PTHrP mRNA content (RT-PCR). Since ventricular TGF-β1 expression increases under pathophysiological conditions like arterial hypertension, ventricular PTHrP expression was further determined in aging spontaneously hypertensive (SHR-SP) and normotensive rats. TGF-β1 expression was increased in SHR-SP and ventricular PTHrP mRNA expression was downregulated at the age of 10 months. PTHrP expression did not recover in elder SHR-SP in which TGF- β1expression was normalized again. Finally, we investigated ventricular PTHrP expression in rats after banding of the ascending aorta which generates a pressure induced hypertrophy without an induction of TGF- β1expression. In ventricles from these animals, PTHrP expression was transiently increased and normalized at day 3. In conclusion, PTHrP expression was reduced under all conditions in which coronary endothelial cells were exposed to TGF- β1. PTHrP expression does not correlate with cardiac hypertrophy. Since coronary endothelial cells represent the majority of PTHrP producing cells in the ventricle its downregulation by TGF- β1seems to be relevant for the paracrine effects of PTHrP.

The perichondrium plays an important role in mediating the effects of TGF-beta1 on endochondral bone formation.

Endochondral bone formation is complex and requires the coordination of signals from several factors and multiple cell types. Thus, chondrocyte differentiation is regulated by factors synthesized by both chondrocytes and cells in the perichondrium. Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in perichondrium/periosteum resulted in increased hypertrophic differentiation in growth plate chondrocytes, suggesting a role for TGF-beta signaling to the perichondrium in limiting terminal differentiation in vivo. Using an organ culture model, we later demonstrated that TGF-beta1 inhibits chondrocyte proliferation and hypertrophic differentiation by two separate mechanisms. Inhibition of hypertrophic differentiation was shown to be dependent on Parathyroid hormone-related peptide (PTHrP) and expression of PTHrP mRNA was stimulated in the perichondrium after treatment with TGF-beta1. In this report, the hypothesis that the perichondrium is required for the effects of TGF-beta1 on growth and/or hypertrophic differentiation in mouse metatarsal organ cultures is tested. Treatment with TGF-beta1 inhibited expression of type X collagen mRNA in metatarsal cultures with the perichondrium intact. In contrast, hypertrophic differentiation as measured by expression of Type X collagen was not inhibited by TGF-beta1 in perichondrium-free cultures. TGF-beta1 added to intact cultures inhibited BrdU incorporation in chondrocytes and increased incorporation in the perichondrium; however, TGF-beta1 treatment stimulated chondrocyte proliferation in metatarsals from which the perichondrium had been enzymatically removed. These results suggest that the TGF-beta1-mediated regulation of both chondrocyte proliferation and hypertrophic differentiation is dependent upon the perichondrium. Thus, one or several factors from the perichondrium might mediate the way chondrocytes respond to TGF-beta1.

Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation.

Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-beta in limiting terminal differentiation in vivo. Parathyroid hormone-related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-beta and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-beta inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-beta1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-beta in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-beta acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-beta has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.

1,25-dihydroxyvitamin D3 inhibits parathyroid hormone-related peptide mRNA expression in fetal rat long bones in culture.

When fetal rat long bones are incubated in the presence of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], steady-state parathyroid hormone-related peptide (PTHrP) mRNA levels are decreased. This decrease is temporary: it is observed as soon as after 3 h of exposure and reaches a nadir after 6 h. At that time, PTHrP mRNA levels are significantly lower in the experimental than in the control bones. However the inhibitory effect vanishes after 24 h, despite continuous exposure to 1,25(OH)2D3 for even 48 h. This is the first report showing that PTHrP mRNA expression can be regulated in rat fetal long bones in vitro by 1,25(OH)2D3.

Effects of PTH on PTHrP Gene Expression in Human Osteoblasts: Up-Regulation with the Kinetics of an Immediate Early Gene

Parathyroid hormone-related peptide (PTHrP) is a local regulator of human bone turnover, which shares some sequence homology with the systemic hormone parathyroid hormone (PTH). The two proteins exert cellular effects through interaction with a common G protein-coupled PTH/PTHrP receptor on target cells. Whilst the PTH gene has a relatively simple structure the PTHrP gene is complex, alternative splicing of which can generate multiple mRNA species encoding PTHrP of 139, 141 and 173 amino acids. To date little is known regarding the extent to which PTH and PTHrP interact to modulate bone cell function. In this study we have used the quantitative technique of Real-Time polymerase chain reaction (PCR) to investigate the ability of PTH to induce PTHrP expression in SaOS-2 cells and in primary human osteoblasts. In addition, we have used the semi-quantitative techniques of PCR followed by Southern analysis and scanning densitometry to investigate the effects of PTH(1-34) on expression of mRNA species encoding the three PTHrP isoforms. We report a 50 fold increase in PTHrP mRNA expression 30 min after treatment with 100 ng/ml human recombinant PTH (1-34) in SaOS-2 cells, and a 38 fold rise in human osteoblasts 45-90 min post-PTH treatment. mRNA species encoding for PTHrP 1-139, 1-141 and 1-173 were all induced in human osteoblasts 45 min after exposure to PTH. Whilst the 1-139 mRNA species exhibited a sustained expression, both the 1-141 and 1-173 isoforms showed a biphasic induction with a second peak 6 hr post PTH treatment. These data demonstrate that PTH induces expression of the PTHrP gene in both SaOS-2 and primary human osteoblasts with the kinetics of an immediate early gene. Up-regulation of the PTHrP gene in response to PTH may be an important physiological mechanism by which this systemic factor effects a localised response in bone.

Expression of parathyroid hormone-related peptide (PTHrP) in gastric tumours.

Parathyroid hormone-related peptide (PTHrP) is produced by various neoplasms. It has been suggested that it acts as a cytokine for cell proliferation and tumour progression. The purpose of this study was to evaluate PTHrP expression in gastric cancers by immunohistochemistry. PTHrP was expressed in 71 of 92 (77.2 per cent) gastric adenocarcinomas without humoral hypercalcaemia. In contrast, one case (5 per cent) out of 20 adenomas and none of the background non-neoplastic epithelium showed PTHrP immunoreactivity. In carcinomas, PTHrP immunoreactivity was higher in moderately differentiated adenocarcinomas (21/22; 95-5 per cent) and poorly differentiated adenocarcinomas (34/34; 100 per cent) than in well-differentiated adenocarcinomas (10/23; 43 per cent). Furthermore, PTHrP expression was more intense in the deeply invasive portions than in the mucosal carcinomas. High percentages of metastatic tumour cells in regional lymph nodes were immunopositive. PTHrP mRNA expression was confirmed by in situ hybridization in gastric adenocarcinomas. Reverse transcription-polymerase chain reaction (RT-PCR) studies of normal gastric mucosa and four human gastric cancer cell lines detected PTHrP transcription in NUGC-1 (poorly differentiated) and NUGC-3 (poorly differentiated) but not in normal gastric mucosa, MKN-1 (well differentiated), and KATO-III (signet ring cell). These findings suggest that overexpression of PTHrP may be involved in the malignant transformation and progression of gastric carcinomas.

O11. Aetiology of rickets in Nigeria

Parathyroid hormone-related peptide (PTHrP) has been shown to have anabolic effects on bone in women with postmenopausal osteoporosis. On the cellular level PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The calcium concentration is considerably higher in the vicinity of resorbing osteoclasts than in the plasma. Therefore the osteoblasts are likely to be confronted by elevated extracellular calcium concentrations in the areas of resorptive activity. The present study was designed to assess the possibility that extracellular calcium could regulate PTHrP expression in osteoblastic cells. Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The PTHrP release into the culture media was measured by an immunoradiometric assay and the expression of PTHrP, osteocalcin and Runx2 mRNA was assayed by real-time PCR. Increasing the extracellular calcium from 1 mM to 5 mM for 24 h resulted in a 4–6-fold increase in the PTHrP release. PTHrP mRNA was also increased by elevated calcium levels. The effect of calcium stimulation on PTHrP release could be seen within 60 min of treatment. The extracellular calcium sensing receptor (CaR) agonist neomycin mimicked the effects of calcium and the MEK/MAPK inhibitor PD98059 abolished the effect of calcium and neomycin. High extracellular calcium increased the mineralization of hMSC and the expression of osteocalcin, but this effect was not mimicked by neomycin. Our results show that in hMSC, elevated extracellular calcium levels increases both released PTHrP and PTHrP mRNA expression. The effect of calcium on PTHrP can be mimicked by activation of the CaR and can be diminished by inhibition of the MAPK signalling pathway.

Parathyroid hormone related peptide mRNA expression during murine postimplantation development: evidence for involvement in multiple differentiation processes.

In this study we describe the spatio-temporal expression of Parathyroid Hormone related Peptide (PTHrP) mRNA during murine postimplantation development from day 5.5 post coitum (pc) until day 12.5 pc. From day 5.5 pc and onwards PTHrP mRNA was detected in the trophoblast. In addition, at day 5.5 and 6.5 pc epithelial cells of the antimesometrial crypt and cells of the inner zone of the decidua directly adjacent to the implanted embryo expressed PTHrP mRNA. This supported a previous model in which parietal endoderm formation is regulated by a paracrine mechanism involving PTHrP expressing trophoblast cells and receptor expressing extra-embryonal endoderm cells. The first embryonal PTHrP mRNA expression was detected in the roof of the hindbrain at gestation day 10.5 pc. From day 11.5 pc and onwards PTHrP mRNA was detected in the otic vesicle, the semilateral channels, the roof of the hindbrain and later in the choroid plexus, in epithelial cells of the lung and heart ventricle, mesenchymal cells lining the nasal pit, the dermis of the snout and at all sites of endochondral bone formation. The widespread expression of PTHrP mRNA during embryogenesis in extra-embryonic and embryonic tissues suggests the involvement of the peptide in multiple growth and differentiation processes.

Evidence that implicates the parathyroid hormone-related peptide in vascular stenosis. Increased gene expression in the intima of injured carotid arteries and human restenotic coronary lesions.

Proliferation of vascular smooth muscle cells (VSMCs) is considered to be one key event underlying the pathophysiology of restenosis after angioplasty. The parathyroid hormone-related peptide (PTHrP) and its receptor, a local autocrine and paracrine regulator of cellular growth in a variety of normal cell types, have been reported in the vicinity of VSMCs. To investigate how PTHrP might be involved in the process of neointimal formation after balloon angioplasty, we examined PTHrP expression in balloon-denuded rat carotid arteries and human coronary arteries that had been retrieved by directional atherectomy. In rat carotid arteries, the RNase protection assay and in situ hybridization demonstrated that PTHrP mRNA expression increased fourfold to sixfold 1 to 7 days after denudation and continued for 28 days, coincident with downregulation of PTH/PTHrP receptor mRNA expression. In situ hybridization and immunohistochemistry revealed that PTHrP expression in balloon-denuded carotid arteries was mainly localized to the neointima. To confirm the involvement of the PTHrP in human coronary artery restenotic lesions, immunohistochemical analysis of human coronary atherectomy specimens (23 primary and 10 restenotic lesions) was then performed. The number of intimal cells that expressed PTHrP protein was significantly higher in restenotic (407 +/- 53 cells/mm2; range, 143 to 739) than in stable angina (50 +/- 12 cells/mm2; range, 18 to 132; P<.05) or unstable angina (129 +/- 16 cells/mm2; range, 21 to 232; P<.05) specimens. These data demonstrate that PTHrP gene expression in VSMCs markedly increases during neointimal formation, supporting the hypothesis that PTHrP may play an important role in vascular stenosis as a regulator of VSMC proliferation.

Synergistic stimulation of parathyroid hormone-related peptide gene expression by mechanical stretch and angiotensin II in rat aortic smooth muscle cells.

Repetitive cyclic stretch (60 cycles/min) of rat aortic smooth muscle cells dramatically enhances the effect of angiotensin II (AII) on mRNA expression of the vasorelaxant, parathyroid hormone-related peptide (PTHrP). Thus, combined stimulation of rat aortic smooth muscle cells by cyclic stretch and low concentrations of AII, but not either alone, induces a synergistic, marked increase in the PTHrP mRNA level, in a manner dependent on the strength of stretch. This response is accompanied by a synergistic increase in secretion of PTHrP from smooth muscle cells. Removal of extracellular Ca2+ or addition of Ca2+ channel blockers, including Gd3+ and nitrendipine, does not considerably reduce the combined effects of stretch and AII, indicating that this response is not dependent on stretch-induced Ca2+ influx across the plasma membrane. The combined effect of stretch and AII on PTHrP mRNA expression is strongly attenuated by the protein kinase C (PKC) inhibitor staurosporine or by down-regulation of PKC, suggesting that PKC plays an important role in the synergistic response. However, stretch neither elicits activation of phospholipase C or PKC by itself, nor does it enhance AII-induced activation of these enzymes. These results indicate that in vascular smooth muscle cells mechanical stretch acts together with the vasoconstrictor AII to regulate the expression of the vasodilator PTHrP and suggest the role of PTHrP as a local modulator of myogenic tone.

Regulation of parathyroid hormone-related peptide gene expression by estrogen in GH4C1 rat pituitary cells has the pattern of a primary response gene.

The parathyroid hormone-related peptide (PTHrP) gene has been reported to be subject to a wide variety of physiological and pharmacological controls. Two distinct patterns of PTHrP mRNA response have been recognized, one characterized by a prolonged or plateau response lasting many hours to days and the second characterized by rapid induction-deinduction kinetics and lasting 1 to several hours. The kinetics of the second pattern are similar to those displayed by primary response genes like nuclear protooncogenes, cytokines, and growth factors. In GH4C1 rat pituitary cells, 17 beta-estradiol induced a rapid and transient increase in PTHrP mRNA expression, with a peak response at 1-2 h. This response appeared to be due to a rapid and transient burst in gene transcription, which by runoff analysis was maximal at 20-40 min and declined thereafter. PTHrP mRNA half-life was 30 min in these cells and was unaltered by estradiol. Cycloheximide did not block the 17 beta-estradiol-induced response but rather prolonged it, and runoff analysis revealed that this effect was due to a prolongation or persistence of PTHrP gene transcription. These findings suggest that the transient nature of the native response reflects the effects of an estrogen-inducible repressor. All of these features are characteristic of a prototypical primary response gene.

Region-specific methylation of the parathyroid hormone-related peptide gene determines its expression in human renal carcinoma cell lines.

Tumor production of a parathyroid hormone-related peptide (PTHrP) is a common cause of the syndrome of humoral hypercalcemia of malignancy, which is frequently associated with renal cell carcinomas. Why certain renal cell carcinomas produce PTHrP while others do not is unknown. Using a system of 12 human renal carcinoma cell lines which either do (n = 6) or do not (n = 6) produce PTHrP, we found that the expression of the PTHrP gene in these cell lines is controlled at the transcriptional level. Transfection studies failed to demonstrate variation in PTHrP promoter activity in these cell lines sufficient to account for the differential PTHrP expression, implicating a cis-acting mechanism. Transcription of the PTHrP gene in these cell lines was found to correlate with the methylation state of specific CpG dinucleotides located within the promoter region but outside a CpG island. The functional importance of this mechanism of control was confirmed by the ability of the demethylating agent, 5-azacytidine, to induce PTHrP mRNA expression in previously nonexpressing cell lines.