Parathyroid Hormone-related Protein mRNA Research Articles

Cyclooxygenase-2 regulates PTHrP transcription in human articular chondrocytes and is involved in the pathophysiology of osteoarthritis in rats.

BackgroundCyclooxygenase-2 (COX-2) inhibitors are prescribed for the management of osteoarthritis (OA)-associated pain and inflammation. However, the role of COX-2 in normal and osteoarthritic articular chondrocytes has not been well investigated. We hypothesize that COX-2 plays a role in articular chondrocytes under normal conditions and during OA progression.MethodsIn vivo COX-2 levels in articular cartilage of normal and papain-induced osteoarthritic rats were compared. The role of COX-2 in human articular chondrocytes (HACs) was tested in vitro by COX-2 overexpression or activity inhibition. The levels of COX-2 and marker gene for normal function or articular cartilage degeneration were evaluated: mRNA by qRT-PCR; proteins by western blotting or immunohistochemistry; and glycosaminoglycan (GAG) by Safranin O–fast green staining. Parathyroid hormone-related protein (PTHrP) promoter activity was detected with luciferase reporter assays.ResultsIn the OA rat study, COX-2 and PTHrP were simultaneously increased in osteoarthritic rat chondrocytes, while increased PTHrP levels were reduced by celecoxib, a COX-2 selective inhibitor. The levels of normal cartilage matrices, GAG and type II collagen decreased, while markers of degeneration, collagen type X and MMP13 were elevated in osteoarthritic articular chondrocytes. Celecoxib rescued the loss of GAG and the increased collagen type X and MMP13 levels. In vitro, COX-2 overexpression in HACs significantly increased Col2a1, Col10a1, PTHrP and MMP13 mRNA expression, which was decreased when COX-2 activity was suppressed. More importantly, COX-2 overexpression upregulated the PTHrP transcription, mRNA expression and protein levels.ConclusionCOX-2 plays a pathophysiological role by preventing terminal differentiation of articular chondrocytes by upregulating PTHrP expression at the early stage of OA progression.The Translational potential of this articleCOX2 up-regulates PTHrP expression in normal and OA articular chondrocytes.

SAT-009 SSRI Use in the Peripartum Period Regulates Mammary Gland Parathyroid Hormone Related Protein (PTHrP) by a Serotonylation-Dependent Mechanism

During lactation, a woman experiences a considerable amount of bone loss and recent studies suggest bone deficits persist years postpartum. Furthermore, selective serotonin uptake inhibitors (SSRIs), which are often prescribed to women experiencing peripartum depression, have been linked to osteopenia. Serotonin signaling can increase parathyroid hormone related protein (PTHrP), a bone remodeling protein which liberates calcium for the milk. Additionally, fluoxetine (a common SSRI) results in increased mammary gland serotonin content and PTHrP, and treatment during the peripartal period reduced maternal bone mineral density. One proposed mechanism of serotonin action is by its covalent addition to proteins by transglutaminase (TG2), termed serotonylation. We therefore investigated whether the combination of fluoxetine and lactation can exacerbate maternal bone loss and the underlying mechanism. We hypothesized that SSRI-induced serotonin signaling in the lactating mammary gland increases PTHrP through a serotonylation-dependent mechanism. Treatment of mouse mammary epithelial cells (HC11) with fluoxetine significantly upregulates PTHrP gene expression and the concentration of its downstream effector, cAMP, over control (P < 0.0004). Furthermore, treatment of the HC11 cells with fluoxetine in addition to a TG2 inhibitor, monodansylcadaverine, restores PTHrP mRNA expression to levels observed in the control. Small g-proteins have emerged as a common target protein for serotonylation. Currently, our data suggest that the g-proteins, RhoA and Rab4, are potential serotonylation targets in the mammary gland. Together these data suggest that the molecular process of serotonyation in HC11 cells links serotonin signaling to increased PTHrP expression. Future work is directed at using the cre-lox system to genetically ablate serotonylation using a WAPCre/TG2Flox transgenic mouse to determine whether decreasing serotonylation in vivo in the mammary gland during lactation improves maternal bone mass.

Loss of Nuclear Localized Parathyroid Hormone-Related Protein in Primary Breast Cancer Predicts Poor Clinical Outcome and Correlates with Suppressed Stat5 Signaling.

Parathyroid hormone-related protein (PTHrP) is required for normal mammary gland development and biology. A PTHLH gene polymorphism is associated with breast cancer risk, and PTHrP promotes growth of osteolytic breast cancer bone metastases. Accordingly, current dogma holds that PTHrP is upregulated in malignant primary breast tumors, but solid evidence for this assumption is missing. We used quantitative IHC to measure PTHrP in normal and malignant breast epithelia, and correlated PTHrP levels in primary breast cancer with clinical outcome. PTHrP levels were markedly downregulated in malignant compared with normal breast epithelia. Moreover, low levels of nuclear localized PTHrP in cancer cells correlated with unfavorable clinical outcome in a test and a validation cohort of breast cancer treated at different institutions totaling nearly 800 cases. PTHrP mRNA levels in tumors of a third cohort of 737 patients corroborated this association, also after multivariable adjustment for standard clinicopathologic parameters. Breast cancer PTHrP levels correlated strongly with transcription factors Stat5a/b, which are established markers of favorable prognosis and key mediators of prolactin signaling. Prolactin stimulated PTHrP transcript and protein in breast cancer cell lines in vitro and in vivo, effects mediated by Stat5 through the P2 gene promoter, producing transcript AT6 encoding the PTHrP 1-173 isoform. Low levels of AT6, but not two alternative transcripts, correlated with poor clinical outcome. This study overturns the prevailing view that PTHrP is upregulated in primary breast cancers and identifies a direct prolactin-Stat5-PTHrP axis that is progressively lost in more aggressive tumors.

FOXA2 PROMOTES PROSTATE CANCER BONE COLONIZATION

BackgroundIt is estimated that 26,000 men will die from prostate cancer (PCa) in 2017. Androgen deprivation therapy (ADT) is the gold standard treatment for advanced PCa. Initially, patients respond to treatment, however, these tumors almost invariably progress to castrate‐resistant PCa, for which there is no cure. Most PCa patients who fail ADT develop metastasis and preferentially relocate to the bones. Bone metastasis results in significant morbidity and mortality as the average time to death is approximately 3–5 years with no treatment available. Understanding the mechanisms by which PCa grow in the bone is critical for the development of novel therapeutics to treat and decrease tumor‐mediated bone destruction.MethodsGene expression profiling studies have found that FOXA2, a forkhead transcription factor that is expressed in embryonic prostate and neuroendocrine PCa, is present in a subset of metastatic PCa specimens. Our preliminary study found FOXA2 expression in a sample set of human PCa bone metastases, suggesting an involvement of FOXA2 in human PCa bone metastases. Also, we found high levels of FOXA2 in aggressive PCa PC3 cells, but not in PCa LNCaP cells. PC3 cells generate osteolytic lesions in bone, whereas LNCaP cells minimally grow following bone inoculation. To establish FOXA2's role in promoting PCa metastasis, FOXA2 was stably knocked down in PC3 cells and overexpressed in LNCaP cells.ResultsWe found that FOXA2 knockdown in PC3 cells resulted in a significant decrease in PC3 mediated in vivo bone destruction following intra‐tibial injection. To understand how FOXA2 is facilitating these changes, we examined the expression of integrins and observed that FOXA2 knockdown decreased the expression of collagen‐binding integrins α1 and αv in PC3 cells. Furthermore, we found FOXA2 knockdown decreased PC3 cells' adhesion and spreading on collagen I (a major component of bone ECM) coated surfaces. The Foxa2‐controlled expression of integrins α1 and αv and the resulting changes in the adherence and spreading would provide a mechanism for PCa cells colonize bone and initiate the bone‐destruction cycle. Additionally, FOXA2 knockdown in PC3 cells resulted in a significant decrease in expression of parathyroid hormone‐related protein (PTHrP), a bone remodeling‐associated protein. We observed that PTHrP mRNA was decreased in FOXA2 knockdown cells and protein in FOXA2 knockdown subcutaneous grafts. When PC3 FOXA2 knockdown cells were co‐cultured with osteoblast and osteoclast, osteoclast markers were decreased.ConclusionsTaken together, FOXA2 plays a major role in facilitating PCa's ability to colonize bone, and further, promote osteoclast activation.Support or Funding InformationFeist‐Weiller Cancer Center/Foundation of Legacy Funds The Office of Research SEED awardsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Wnt signaling induces gene expression of factors associated with bone destruction in lung and breast cancer.

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone destruction in bone metastatic tumors. Transforming growth factor-beta (TGF-β) stimulates PTHrP production in part through the transcription factor Gli2, which is regulated independent of the Hedgehog signaling pathway in osteolytic cancer cells. However, inhibition of TGF-β in vivo does not fully inhibit tumor growth in bone or tumor-induced bone destruction, suggesting other pathways are involved. While Wnt signaling regulates Gli2 in development, the role of Wnt signaling in bone metastasis is unknown. Therefore, we investigated whether Wnt signaling regulates Gli2 expression in tumor cells that induce bone destruction. We report here that Wnt activation by β-catenin/T cell factor 4 (TCF4) over-expression or lithium chloride (LiCl) treatment increased Gli2 and PTHrP expression in osteolytic cancer cells. This was mediated through the TCF and Smad binding sites within the Gli2 promoter as determined by promoter mutation studies, suggesting cross-talk between TGF-β and Wnt signaling. Culture of tumor cells on substrates with bone-like rigidity increased Gli2 and PTHrP production, enhanced autocrine Wnt activity and led to an increase in the TCF/Wnt signaling reporter (TOPFlash), enriched β-catenin nuclear accumulation, and elevated Wnt-related genes by PCR-array. Stromal cells serve as an additional paracrine source of Wnt ligands and enhanced Gli2 and PTHrP mRNA levels in MDA-MB-231 and RWGT2 cells in vitro and promoted tumor-induced bone destruction in vivo in a β-catenin/Wnt3a-dependent mechanism. These data indicate that a combination of matrix rigidity and stromal-secreted factors stimulate Gli2 and PTHrP through Wnt signaling in osteolytic breast cancer cells, and there is significant cross-talk between the Wnt and TGF-β signaling pathways. This suggests that the Wnt signaling pathway may be a potential therapeutic target for inhibiting tumor cell response to the bone microenvironment and at the very least should be considered in clinical regimens targeting TGF-β signaling.

Deletion of the nuclear localization sequences and C-terminus of PTHrP impairs embryonic mammary development but also inhibits PTHrP production.

Parathyroid hormone-related protein (PTHrP) can be secreted from cells and interact with its receptor, the Type 1 PTH/PTHrP Receptor (PTHR1) in an autocrine, paracrine or endocrine fashion. PTHrP can also remain inside cells and be transported into the nucleus, where its functions are unclear, although recent experiments suggest that it may broadly regulate cell survival and senescence. Disruption of either the PTHrP or PTHR1 gene results in many abnormalities including a failure of embryonic mammary gland development in mice and in humans. In order to examine the potential functions of nuclear PTHrP in the breast, we examined mammary gland development in PTHrP (1–84) knock-in mice, which express a mutant form of PTHrP that lacks the C-terminus and nuclear localization signals and which can be secreted but cannot enter the nucleus. Interestingly, we found that PTHrP (1–84) knock-in mice had defects in mammary mesenchyme differentiation and mammary duct outgrowth that were nearly identical to those previously described in PTHrP−/− and PTHR1−/− mice. However, the mammary buds in PTHrP (1–84) knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function. Examination of the effects of nuclear PTHrP in the mammary gland in vivo will require the development of alternative animal models.

Contribution of PTHrP to mechanical strain-induced fibrochondrogenic differentiation in entheses of Achilles tendon of miniature pigs

BackgroundFibrochondrocytes are involved in entheses repair, but their response to mechanical strain (MS) is ill known. ObjectiveTo determine if parathyroid hormone-related protein (PTHrP) expression in fibrochondrocytes from fibrocartilaginous entheses is modulated by MS, and to further observe the regulatory effects of human (h) PTHrP on fibrochondrocyte differentiation. MethodsFibrochondrocytes from fibrocartilaginous entheses of Guizhou miniature pig׳s Achilles tendon were submitted or not to MS (4%, 8% or 12% cyclic tensile strain; 1Hz). Fibrochondrocytes were also exposed to: cyclopamine (Indian hedgehog (Ihh) inhibitor) (10μM), hPTHrP (10nM) or cyclopamine/hPTHrP (cyclopamine 10uM+hPTHrP 10nM). Types I, II and X collagen and PTHrP expressions were measured by real-time RT-PCR and Western blot. ResultUnder 4% strain load for 12h, types I and II collagen mRNA expressions were increased (+324% and +659%, P<0.001), while type X collagen was decreased (−89%, P<0.001). At 12%, types I and II collagen mRNA expressions were decreased (−62% and −62%, P<0.001), while type X collagen was increased (+375%, P<0.05). Under 4% strain load, PTHrP mRNA expression was increased in relation with strain duration (from 3 to 12h: +168%, P<0.001), while at 12%, PTHrP expression decreased with time (from 3 to 12h: −81%, P<0.001). Using cyclopamine for 24h, PTHrP mRNA expression was significantly decreased (−88%, P<0.05), types I and II collagen were decreased (−90% and −82%, P<0.001), and type X collagen was increased (+261%, P<0.001). ConclusionsDynamic MS modulate PTHrP expressions. Thus, PTHrP might play an important role in fibrochondrocyte differentiation, indirectly revealing a role in entheses׳ formation and repair.

Cinacalcet attenuates hypercalcemia observed in mice bearing either Rice H-500 Leydig cell or C26-DCT colon tumors

Excessive secretion of parathyroid hormone-related protein (PTHrP) by tumors stimulates bone resorption and increases renal tubular reabsorption of calcium, resulting in hypercalcemia of malignancy. We investigated the ability of cinacalcet, an allosteric modulator of the calcium-sensing receptor, to attenuate hypercalcemia by assessing its effects on blood ionized calcium, serum PTHrP, and calcium-sensing receptor mRNA in mice bearing either Rice H-500 Leydig cell or C26-DCT colon tumors. Cinacalcet effectively decreased hypercalcemia in a dose- and enantiomer-dependent manner; furthermore, cinacalcet normalized phosphorus levels, but did not affect serum PTHrP. Ribonuclease protection assay results demonstrated presence of PTHrP receptor, but not calcium-sensing receptor mRNA in C26-DCT tumors. The mechanism by which cinacalcet lowered serum calcium was investigated in parathyroidectomized rats (i.e., without PTH) made hypercalcemic by PTHrP. Cinacalcet attenuated PTHrP-mediated elevations in blood ionized calcium, which were accompanied by increased plasma calcitonin. Taken together these results suggest that the cinacalcet-mediated decrease in serum calcium is not the result of a direct effect on tumor cells, but rather is the result of increased calcitonin release. In summary, cinacalcet effectively reduced tumor-mediated hypercalcemia and corrected hypophosphatemia in mice. Further investigation of cinacalcet for treatment of hypercalcemia of malignancy is warranted.

PTHrP is a novel mediator for TGF-β-induced apoptosis

Parathyroid hormone-related protein (PTHrP) is a polyhormone secretory protein that plays fundamental roles in the development and function of various tissues. Transforming growth factor (TGF)-β is an important tumor suppressor that induces cell cycle arrest and apoptosis. Increased PTHrP expression has been implicated in TGF-β-induced growth inhibition in human hepatocellular carcinoma cells. However, whether PTHrP is involved in TGF-β-induced apoptosis remains unknown. Using Hep3B and HuH-7, two human hepatocellular carcinoma cell lines, the current study examined the hypothesis that TGF-β-induced apoptosis is mediated by the induction of PTHrP expression. We found that (1) TGF-β induces PTHrP mRNA expression, protein expression and secretion in a time-dependent fashion; (2) knockdown of PTHrP gene expression or neutralization of secreted PTHrP isoforms blocks TGF-β-induced apoptosis; and (3) TGF-β-induced PTHrP expression is Smad3-dependent. Thus, we have identified PTHrP as a novel mediator for TGF-β-induced apoptosis in Hep3B cells. Our findings provide further insights into the mechanisms through which TGF-β conveys tumor suppression activity.

Construction of recombinant adenoviruses carrying the optimal shRNA template against goat PTHrP and successful suppression of PTHrP expression in mammary epithelial cells

Parathyroid hormone-related protein (PTHrP) is a protein member of the parathyroid hormone family that regulates the dynamic balance between blood and bone calcium during lactation. However, the mechanism of its regulation is not very clear. In order to establish a framework for further functional studies of the PTHrP gene in goat mammary gland epithelial cells during the lactation period, PTHrP cDNA was isolated from Xinong Saanen dairy goats. Its coding sequence is 534 bp in size. We also designed a short hairpin RNA (shRNA) to efficiently inhibit PTHrP expression and constructed recombinant adenoviruses carrying a template encoding this shRNA (AD-PTHrP-322) using the Block-iT shRNA interference system. Finally, the inhibition of PTHrP expression by the recombinant adenoviruses was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blotting. qRT-PCR results showed that the expression of PTHrP mRNA in mammary epithelial cells was downregulated by 29.2, 68.1, and 82.6% 24, 48, and 72 h after the cells were infected with AD-PTHrP-322, respectively. Western blotting also showed that the expression of PTHrP was reduced in a time-dependent manner. These results suggest that AD-PTHrP-322 significantly inhibits the expression of PTHrP.

Myeloid-derived suppressor cells expand during breast cancer progression and promote tumor-induced bone destruction

Myeloid-derived suppressor cells (MDSCs), identified as Gr1+CD11b+ cells in mice, expand during cancer and promote tumor growth, recurrence and burden. However, little is known about their role in bone metastases. We hypothesized that MDSCs may contribute to tumor-induced bone disease, and inoculated breast cancer cells into the left cardiac ventricle of nude mice. Disease progression was monitored weekly by X-ray and fluorescence imaging and MDSCs expansion by fluorescence-activated cell sorting. To explore the contribution of MDSCs to bone metastasis, we co-injected mice with tumor cells or PBS into the left cardiac ventricle and Gr1+CD11b+ cells isolated from healthy or tumor-bearing mice into the left tibia. MDSCs didn’t induce bone resorption in normal mice, but increased resorption and tumor burden significantly in tumor-bearing mice. In vitro experiments showed that Gr1+CD11b+ cells isolated from normal and tumor-bearing mice differentiate into osteoclasts when cultured with RANK ligand and macrophage colony-stimulating factor, and that MDSCs from tumor-bearing mice upregulate parathyroid hormone-related protein (PTHrP) mRNA levels in cancer cells. PTHrP upregulation is likely due to the 2-fold increase in transforming growth factor β expression that we observed in MDSCs isolated from tumor-bearing mice. Importantly, using MDSCs isolated from GFP-expressing animals, we found that MDSCs differentiate into osteoclast-like cells in tumor-bearing mice as evidenced by the presence of GFP+TRAP+ cells. These results demonstrate that MDSCs expand in breast cancer bone metastases and induce bone destruction. Furthermore, our data strongly suggest that MDSCs are able to differentiate into osteoclasts in vivo and that this is stimulated in the presence of tumors.

AU-rich elements in the 3′-UTR regulate the stability of the 141 amino acid isoform of parathyroid hormone-related protein mRNA

We demonstrated previously that parathyroid hormone-related protein (PTHrP) 1-141 mRNA is the least stable of three isoforms and is the only isoform that is stabilized by TGF-β. In order to understand how PTHrP mRNA is stabilized by TGF-β, we first sought to elucidate the mechanism(s) that are responsible for the instability of PTHrP isoform 1-141 mRNA. The 3′-UTR of isoform 1-141 contains four AU-rich elements (AREs), which are known to mediate mRNA degradation. We utilized a luciferase reporter system to test whether these four AREs are responsible for the short half-life of PTHrP 1-141 mRNA. Our results demonstrated that ARE elements in the 3′-UTR of PTHrP 1-141 mRNA play a significant role in regulation of the stability of the mRNA. It is known that AREs mediate their effects on mRNA stability through a number of ARE-binding proteins that recruit the exosome, a complex of exonucleases that degrades the mRNA. We identified tristetraproline (TTP) as an RNA-binding protein that may be involved in ARE-mediated degradation of PTHrP 1-141 mRNA.

Methylation of specific CpG sites in the P2 promoter of parathyroid hormone-related protein determines the invasive potential of breast cancer cell lines

Parathyroid hormone-related protein (PTHrP) is upregulated in primary breast cancers and a major candidate for osteoclastic bone resorption present at sites of breast cancer to bone metastases. Using a human model of mammary epithelial cell lines differing in tumorigenicity and PTHrP expression, we investigated the role of epigenetic modifications for PTHrP expression. Quantitative analysis of the DNA methylation patterns at a total of 104 CpGs in the promoter region of PTHrP by pyrosequencing showed the absence of methylation in all analyzed cell lines in the large CpG island upstream of exon 1C. In the second intron of promoter 2 (P2) a region was identified containing 4 CpG nucleotides for which differential methylation correlated with the PTHrP expression level. The functional importance of this control mechanism was confirmed by the ability of the demethylating agent 5'-azacytidine to induce PTHrP mRNA and iPTHrP protein expression in previously non-expressing cell lines and increase their production by metastatic NS2T2A1 cells. In particular, transcription from P2 was activated non-tumoral S1T3 cells upon treatment with 5'-azacytidine. Our findings support the hypothesis that the methylation status of specific CpG dinucleotides is the dominant mechanism involved in silencing of PTHrP expression rather than the overall methylation of the CpG island. Methylation of the PTHrP P2 is a potential marker of breast cancer progression and might be used to evaluate the metastatic potential of breast tumors.

1,25-Dihydroxyvitamin D 3 regulates PTHrP expression via transcriptional, post-transcriptional and post-translational pathways

Parathyroid hormone-related protein (PTHrP) increases the growth and osteolytic potential of prostate cancer cells, making it important to control PTHrP expression. PTHrP expression is suppressed by 1,25-dihydroxyvitamin D 3 (1,25D). The aim of this study was to identify the pathways via which 1,25D exerts these effects. Our main findings are that 1,25D regulates PTHrP levels via multiple pathways in PC-3 and C4-2 (human prostate cancer) cell lines, and regulation is dependent on VDR expression. The human PTHrP gene has three promoters (P); PC-3 cells preferentially utilize P2 and P3, while C4-2 cells preferentially utilize P1. 1,25D regulates PTHrP transcriptional activity from both P1 and P3. The 1,25D-mediated decrease in PTHrP mRNA levels also involves a post-transcriptional pathway since 1,25D decreases PTHrP mRNA stability. 1,25D also suppresses PTHrP expression directly at the protein level by increasing its degradation. Regulation of PTHrP levels is dependent on VDR expression, as using siRNAs to deplete VDR expression negates the 1,25D-mediated downregulation of PTHrP expression. These results indicate the importance of maintaining adequate 1,25D levels and VDR status to control PTHrP levels.

TGF-β Promotion of Gli2-Induced Expression of Parathyroid Hormone-Related Protein, an Important Osteolytic Factor in Bone Metastasis, Is Independent of Canonical Hedgehog Signaling

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-β, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormone-related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 overexpression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-MB-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-repressor gene (Gli2-rep) reduced endogenous and TGF-β-stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep-expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-β propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-β signaling, Gli2 expression was downregulated in cells, whereas enforced overexpression of Gli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-β to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction.

Combinatorial library discovery of small molecule inhibitors of lung cancer proliferation and parathyroid hormone-related protein expression

PTHrP (parathyroid hormone-related protein) is abnormally expressed in a substantial majority of lung cancers, especially non-small cell lung cancers, and plays a key role in tumor progression. Thus, this oncoprotein could be a target for treating patients with lung cancer. This study screened combinatorial libraries of heterocyclic amines for inhibitory effects on PTHrP expression and cell proliferation. Two libraries of over 780,000 bis-cyclic thiourea and guanidine compounds each were tested in BEN lung carcinoma cells. The number of PTHrP inhibitors and the magnitude of the reduction in PTHrP were greater for thioureas. Selected lead thiourea compounds decreased cell PTHrP protein content in dose-dependent fashion, reduced relative abundance of PTHrP mRNA, decreased transcripts derived from the PTHrP P3 promoter and reduced activity of a full length PTHrP promoter luciferase construct. Similar effects on PTHrP mRNA were observed in A549 and H441 lung adenocarcinoma cells and in H727 lung carcinoid cells. However, the compounds only inhibited PTHrP protein levels in BEN cells and H727 cells. The compounds reduced the rate of cell proliferation in BEN cells and H727 cells, but not in lines that showed no inhibition of PTHrP protein. These results suggest that cyclic thiourea compounds inhibit PTHrP expression mediated by the P3 promoter, which is widely used in the majority of PTHrP-expressing cells, and that they may inhibit growth of lung cancer cells through the same mechanism. Further work will be necessary to investigate their mechanism for effects on growth of PTHrP-positive tumors in vivo.

Phosphate-induced Apoptosis of Hypertrophic Chondrocytes Is Associated with a Decrease in Mitochondrial Membrane Potential and Is Dependent upon Erk1/2 Phosphorylation

Growth plate abnormalities, associated with impaired hypertrophic chondrocyte apoptosis, are observed in humans and animals with abnormalities of vitamin D action and renal phosphate reabsorption. Low circulating phosphate levels impair hypertrophic chondrocyte apoptosis, whereas treatment of these cells with phosphate activates the mitochondrial apoptotic pathway. Because phosphate-mediated apoptosis of chondrocytes is differentiation-dependent, studies were performed to identify factors that contribute to hypertrophic chondrocyte apoptosis. An increase in the percentage of cells with low mitochondrial membrane potential, evaluated by JC-1 fluorescence, was observed during hypertrophic differentiation of primary murine chondrocytes in culture. This percentage was further increased by treatment of hypertrophic, but not proliferative, chondrocytes with phosphate. Phosphate-mediated apoptosis was observed as early as 30 min post-treatment and was dependent upon Erk1/2 phosphorylation. Inhibition of Erk1/2 phosphorylation in vivo confirmed an important role for this signaling pathway in regulating hypertrophic chondrocyte apoptosis in growing mice. Murine embryonic metatarsals cultured under phosphate-restricted conditions demonstrated a 2.5-fold increase in parathyroid hormone-related protein mRNA expression accompanied by a marked attenuation in phospho-Erk immunoreactivity in hypertrophic chondrocytes. Thus, these investigations point to an important role for phosphate in regulating mitochondrial membrane potential in hypertrophic chondrocytes and growth plate maturation by the parathyroid hormone-related protein signaling pathway.

E1A oncogene expression inhibits PTHrP P3 promoter activity and sensitizes human prostate cancer cells to TNF-induced apoptosis

In the advanced stages of prostate cancer, tumor cells can evolve to become androgen-independent and resistant to injury-induced apoptosis. Tumor cell expression of parathyroid hormone-related protein (PTHrP) may contribute to the apoptosis phenotype. Expression of the adenovirus E1A oncogene repressed PTHrP promoter and mRNA expression in human PC-3 prostate cancer cells and increased the caspase 3 activation and sensitivity of these cells to apoptosis triggered by tumor necrosis factor alpha. These results suggest that strategies aimed at modulating PTHrP expression may increase the efficacy of innate immune effector mechanisms and proapoptotic, therapeutics in prostate cancer.

Dexamethasone Suppresses Parathyroid Hormone-related Protein Expression by Human Oral Squamous Carcinoma Cells (HSC-3)

We examined the effect of dexamethasone on parathyroid hormone-related protein (PTHrP) mRNA expression and PTHrP production in HSC-3 cells, a human oral squamous carcinoma cell line. Dexamethasone suppressed PTHrP mRNA expression and PTHrP production in a dose-dependent manner. A time course study demonstrated a significant decrease in PTHrP mRNA from as early as 6 h after the initiation of exposure to 10−1 M dexamethasone, and the suppressive effect was stronger at 24 h. Dexamethasone-induced suppression of PTHrP mRNA expression and PTHrP production was abolished in the presence of RU486, a glucocorticoid receptor antagonist. Dexamethasone decreased the transcriptional activity of the PTHrP promoter. These findings show that dexamethasone acts through binding to functional glucocorticoid receptors and down-regulates PTHrP gene expression via a transcriptional mechanism in HSC-3 cells.

The calcium‐sensing receptor and parathyroid hormone‐related protein are expressed in differentiated, favorable neuroblastic tumors

Differentiated histopathology is a favorable prognostic factor in neuroblastic tumors, and molecular pathways underlying neuroblastoma differentiation can be modulated pharmacologically. The calcium-sensing receptor (CaR) and parathyroid hormone-related protein (PTHrP) regulate differentiation processes in some cellular contexts. CaR is up-regulated when neural stem cells are specified to the oligodendrocyte lineage and regulates PTHrP production in astrocytes. The objective of the current study was to assess whether CaR and PTHrP participate in neuroblastoma differentiation pathways. CaR and PTHrP messenger RNA (mRNA) and protein expression were analyzed in neuroblastic tumors, and correlation with prognostic factors was assessed. CaR and PTHrP expression levels were analyzed in neuroblastoma cell lines treated with all-trans-retinoic acid or 5-bromo-2'-deoxyuridine (BrdU). CaR expression was correlated with favorable histology, age at diagnosis <1 year, low clinical stage, and low clinical risk. CaR was absent in undifferentiated neuroblasts and was expressed in differentiating neuroblasts. CaR and PTHrP were highly expressed in ganglion and in Schwann-like cells. PTHrP mRNA levels were higher in ganglioneuroblastomas and ganglioneuromas than in neuroblastomas (P < .0001). Both genes were up-regulated in neuroblastomas with treatment-induced maturation features. CaR, but not PTHrP, was up-regulated at early phases of in vitro neuronal differentiation induction. Substrate-adherent, non-neuronal cell lines displayed the highest PTHrP levels among the neuroblastoma cell lines examined. The up-regulation of PTHrP and of 2 glial differentiation markers was observed in 2 cell lines that were treated with BrdU, whereas CaR was induced in only 1 cell line. CaR and PTHrP were expressed in differentiated, favorable neuroblastic tumors, and both genes were up-regulated by inducing differentiation.