Induction of chronic pancreatitis by pancreatic duct ligation activates BMP2, apelin, and PTHrP expression in mice.

Chronic pancreatitis (CP) is a devastating disease with no treatments. Experimental models have been developed to reproduce the parenchyma and inflammatory responses typical of human CP. For the present study, one objective was to assess and compare the effects of pancreatic duct ligation (PDL) to those of repetitive cerulein (Cer)-induced CP in mice on pancreatic production of bone morphogenetic protein-2 (BMP2), apelin, and parathyroid hormone-related protein (PTHrP). A second objective was to determine the extent of cross talk among pancreatic BMP2, apelin, and PTHrP signaling systems. We focused on BMP2, apelin, and PTHrP since these factors regulate the inflammation-fibrosis cascade during pancreatitis. Findings showed that PDL- and Cer-induced CP resulted in significant elevations in expression and peptide/protein levels of pancreatic BMP2, apelin, and PTHrP. In vivo mouse and in vitro pancreatic cell culture experiments demonstrated that BMP2 stimulated pancreatic apelin expression whereas apelin expression was inhibited by PTHrP exposure. Apelin or BMP2 exposure inhibited PTHrP expression, and PTHrP stimulated upregulation of gremlin, an endogenous inhibitor of BMP2 activity. Transforming growth factor-ß (TGF-ß) stimulated PTHrP expression. Together, findings demonstrated that PDL- and Cer-induced CP resulted in increased production of the pancreatic BMP2, apelin, and PTHrP signaling systems and that significant cross talk occurred among pancreatic BMP2, apelin, and PTHrP. These results together with previous findings imply that these factors interact via a pancreatic network to regulate the inflammation-fibrosis cascade during CP. More importantly, this network communicated with TGF-ß, a key effector of pancreatic pathophysiology. This novel network may be amenable to pharmacologic manipulations during CP in humans.

Apigenin inhibits pancreatic stellate cell activity in pancreatitis.

BACKGROUND: Chronic pancreatitis (CP) is characterized by recurrent pancreatic injury, resulting in inflammation, necrosis, and fibrosis. There are currently no drugs limiting pancreatic fibrosis associated with CP, and there is a definite need to fill this void in patient care. MATERIALS AND METHODS: Pancreatitis was induced in C57/BL6 mice using supraphysiologic doses of cerulein, and apigenin treatment (once daily, 50 µg per mouse by oral gavage) was initiated 1 wk into the recurrent acute pancreatitis (RAP) protocol. Pancreata were harvested after 4 wk of RAP. Immunostaining with fibronectin antibody was used to quantify the extent of pancreatic fibrosis. To assess how apigenin may decrease organ fibrosis, we evaluated the effect of apigenin on the proliferation and apoptosis of human pancreatic stellate cells (PSCs) in vitro. Finally, we assessed apigenin's effect on the gene expression in PSCs stimulated with parathyroid hormone-related protein, a profibrotic and proinflammatory mediator of pancreatitis, using reverse transcription-polymerase chain reaction. RESULTS: After 4 wk of RAP, apigenin significantly reduced the fibrotic response to injury while preserving acinar units. Apigenin inhibited viability and induced apoptosis of PSCs in a time- and dose-dependent manner. Finally, apigenin reduced parathyroid hormone-related protein-stimulated increases in the PSC messenger RNA expression levels of extracellular matrix proteins collagen 1A1 and fibronectin, proliferating cell nuclear antigen, transforming growth factor-beta, and interleukin-6. CONCLUSIONS: These in vivo and in vitro studies provide novel insights regarding apigenin's mechanism(s) of action in reducing the severity of RAP. Additional preclinical testing of apigenin analogs is warranted to develop a therapeutic agent for patients at risk for CP.

Acinar cell-specific knockout of the PTHrP gene decreases the proinflammatory and profibrotic responses in pancreatitis.

Pancreatitis is a necroinflammatory disease with acute and chronic manifestations. Accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic pancreatitis (CP). Pancreatic parathyroid hormone-related protein (PTHrP) levels are elevated in a mouse model of cerulein-induced AP. Here, we show elevated PTHrP levels in mouse models of pancreatitis induced by chronic cerulein administration and pancreatic duct ligation. Because acinar cells play a major role in the pathophysiology of pancreatitis, mice with acinar cell-specific targeted disruption of the Pthrp gene (PTHrP(Δacinar)) were generated to assess the role of acinar cell-secreted PTHrP in pancreatitis. These mice were generated using Cre-LoxP technology and the acinar cell-specific elastase promoter. PTHrP(Δacinar) exerted protective effects in cerulein and pancreatic duct ligation models, evident as decreased edema, histological damage, amylase secretion, pancreatic stellate cell (PSC) activation, and extracellular matrix deposition. Treating acinar cells in vitro with cerulein increased IL-6 expression and NF-κB activity; these effects were attenuated in PTHrP(Δacinar) cells, as were the cerulein- and carbachol-induced elevations in amylase secretion. The cerulein-induced upregulation of procollagen I expression was lost in PSCs from PTHrP(Δacinar) mice. PTHrP immunostaining was elevated in human CP sections. The cerulein-induced upregulation of IL-6 and ICAM-1 (human acinar cells) and procollagen I (human PSCs) was suppressed by pretreatment with the PTH1R antagonist, PTHrP (7-34). These findings establish PTHrP as a novel mediator of inflammation and fibrosis associated with CP. Acinar cell-secreted PTHrP modulates acinar cell function via its effects on proinflammatory cytokine release and functions via a paracrine pathway to activate PSCs.

Parathyroid hormone-related protein regulates integrin α6 and ß4 levels via transcriptional and post-translational pathways.

Parathyroid hormone-related protein (PTHrP) enhances prostate cancer (CaP) growth and metastasis in vivo. PTHrP also increases cell survival and migration, and upregulates pro-invasive integrin α6ß4 expression. We used the human CaP cell lines C4-2 and PC-3 as model systems to study the mechanisms via which PTHrP regulates α6ß4 levels. We report that PTHrP regulates α6 and ß4 levels via a transcriptional pathway; ß4 regulation involves the NF-κB pathway. PTHrP also regulates ß4 levels at the post-translational level. PTHrP inhibits caspase-3 and -7 activities. Post-translational regulation of ß4 by PTHrP is mediated via attenuation of its proteolytic cleavage by these caspases. Since α6 dimerizes with ß4, increased ß4 levels result in elevated α6 levels. Suppressing ß4 using siRNA attenuates the effect of caspase inhibition on apoptosis and cell migration. These results provide evidence of a link between PTHrP, integrin α6ß4 levels as a function of caspase activity, and cell survival and migration. Targeting PTHrP in CaP cancer, thereby reversing the effect on caspase activity and α6ß4 levels, may thus prove therapeutically beneficial.

Apigenin Decreases Acinar Cell Damage in Pancreatitis.

OBJECTIVE: Chronic pancreatitis is the consequence of multiple episodes of recurrent acute pancreatitis (RAP). We hypothesized that apigenin can minimize the sequelae of RAP by limiting acinar cells' proinflammatory signaling pathways. METHODS: AR42J acinar cells were treated in vitro with transforming growth factor ß (TGF-ß), apigenin, and other inhibitors. Dual luciferase reporter assay measured parathyroid hormone-related protein (PTHrP) promoter activity. MAPK/ERK pathway activity was assessed by immunoblotting and in vivo by immunohistochemistry with a cerulein-induced RAP mouse model. Nuclear factor κ B nuclear localization was analyzed in vitro in cells stimulated with tumor necrosis factor α. Primary acini were isolated and treated with cerulein; interleukin 6 messenger RNA was measured comparing PTHrP wild-type and knockout mice. RESULTS: Apigenin and PD98059 each downregulated TGF-ß stimulation of PTHrP P3 promoter activity. In a RAP mouse model, apigenin reduced pERK nuclear localization in acinar cells and preserved acinar cell architecture. Apigenin suppressed tumor necrosis factor α-mediated signaling by decreasing nuclear factor κ B nuclear localization and decreased interleukin 6 messenger RNA levels via a PTHrP-dependent mechanism. CONCLUSIONS: Apigenin reduced inflammatory responses in experimental models of RAP. The mechanisms mediating the actions of apigenin, in part, are owing to attenuation of PTHrP and TGF-ß proinflammatory signaling.

PTHrP increases xenograft growth and promotes integrin alpha6beta4 expression and Akt activation in colon cancer.

Parathyroid hormone-related protein (PTHrP) is expressed by human colon cancer tissue and cell lines. Expression of PTHrP and phosphatidylinositol 3-kinase (PI3-K) pathway components correlates with the severity of colon carcinoma. Here we observed a positive effect of endogenous PTHrP on LoVo (human colon cancer) cell proliferation, migration, invasion, integrin alpha6 and beta4 expression, and p-Akt levels. There was a direct correlation between PTHrP expression and anchorage-independent cell growth. PTHrP significantly increased xenograft growth; tumors from PTHrP-overexpressing cells showed increased expression of integrins alpha6 and beta4, and PI3-K pathway components. The higher expression of PTHrP in human colon cancer adenocarcinoma vs. normal colonic mucosa was accompanied by increased integrin alpha6 and beta4 levels. Elevated PTHrP expression in colon cancer may thus upregulate integrin alpha6beta4 expression, with consequent PI3-K activation. Targeting PTHrP might result in effective inhibition of tumor growth, migration, and invasion.

Increased cell survival, migration, invasion, and Akt expression in PTHrP-overexpressing LoVo colon cancer cell lines.

Parathyroid hormone-related protein (PTHrP) has been localized in human colon cancer tissue and cell lines. We have previously shown that PTHrP increases colon cancer cell proliferation, extracellular matrix adhesion, and cell-surface integrin alpha6beta4 expression. Since cancer cell migration, invasion, and survival are crucial components of metastasis, and colon cancer has a high metastatic potential, in this study we used the human colon cancer cell line LoVo as a model system to study the effects of PTHrP on these parameters. PTHrP expression was modulated by stable transfection with a construct expressing PTHrP (-36 to +139). We report that PTHrP increases cell migration, invasion, and survival. PTHrP altered cell morphology, with PTHrP-overexpressing cells exhibiting increased spreading and several long protrusions. PTHrP also increased the steady-state mRNA levels of the integrin alpha6 and beta4 subunits, indicating a direct and/or indirect effect of PTHrP on the transcriptional and/or post-transcriptional regulation of integrin alpha6 and beta4 expression. Integrin alpha6beta4 activates the phosphoinositol 3-kinase (PI3-K)/Akt pathway, leading to glycogen synthase kinase-3 (GSK-3) deactivation. PTHrP overexpression also led to an increase in active Akt and inactive GSK-3 levels, indicating that the PTHrP-mediated upregulation of integrin alpha6beta4 expression may activate the PI3-K pathway, resulting in increased cell survival, migration and invasion.

PTHrP contributes to the anti-proliferative and integrin alpha6beta4-regulating effects of 1,25-dihydroxyvitamin D(3).

Parathyroid hormone-related protein (PTHrP) increases the growth and metastatic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] suppresses PTHrP expression and exerts an anti-proliferative effect in prostate carcinoma cells. We used the human prostate cancer cell line C4-2 as a model system to ask whether down-regulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the anti-proliferative effects of 1,25(OH)(2)D(3). Since PTHrP increases the expression of the pro-invasive integrin alpha6beta4, we also asked whether 1,25(OH)(2)D(3) decreases integrin alpha6beta4 expression in C4-2 cells, and whether modulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the effects of 1,25(OH)(2)D(3) on integrin alpha6beta4 expression. Two strategies were utilized to modulate PTHrP levels: overexpression of PTHrP (-36 to +139) and suppression of endogenous PTHrP expression using siRNAs. We report a direct correlation between PTHrP expression, C4-2 cell proliferation and integrin alpha6beta4 expression at the mRNA and cell surface protein level. Treatment of parental C4-2 cells with 1,25(OH)(2)D(3) decreased cell proliferation and integrin alpha6 and beta4 expression. These 1,25(OH)(2)D(3) effects were significantly attenuated in cells with suppressed PTHrP expression. 1,25(OH)(2)D(3) regulates PTHrP expression via a negative vitamin D response element (nVDRE) within the noncoding region of the PTHrP gene. The effects of 1,25(OH)(2)D(3) on cell proliferation and integrin alpha6beta4 expression were significantly attenuated in cells overexpressing PTHrP (-36 to +139), which lacks the nVDRE. These findings suggest that one of the pathways via which 1,25(OH)(2)D(3) exerts its anti-proliferative effects is through down-regulation of PTHrP expression.

Prostate cancer cell type-specific involvement of the VDR and RXR in regulation of the human PTHrP gene via a negative VDRE.

Parathyroid hormone-related protein (PTHrP) increases the growth and osteolytic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its non-hypercalcemic analog, EB1089, decrease PTHrP mRNA and cellular protein levels in the androgen-dependent human prostate cancer cell line LNCaP and its androgen-independent derivative, the C4-2 cell line. This effect is mediated via a negative Vitamin D response element (nVDREhPTHrP) within the human PTHrP gene and involves an interaction between nVDREhPTHrP and the Vitamin D receptor (VDR). The retinoid X receptor (RXR) is a frequent heterodimeric partner of the VDR. We show that RXRalpha forms part of the nuclear protein complex that interacts with nVDREhPTHrP along with the VDR in LNCaP and C4-2 cells. We also show that the RXR ligand, 9-cis-retinoic acid, downregulates PTHrP mRNA levels; this decrease is more pronounced in LNCaP than in C4-2 cells. In addition, 9-cis-retinoic acid enhances the 1,25(OH)2D3-mediated downregulation of PTHrP expression in both cell lines; this effect also is more pronounced in LNCaP cells. Proliferation of LNCaP, but not C4-2, cells is decreased by 9-cis-retinoic acid. Promoter activity driven by nVDREhPTHrP cloned upstream of the SV40 promoter and transiently transfected into LNCaP and C4-2 cells is downregulated in response to 1,25(OH)2D3 and EB1089 in both cell lines. Co-treatment with these compounds and 9-cis-retinoic acid further decreases CAT activity in LNCaP, but not C4-2, cells. These results indicate that PTHrP gene expression is regulated by 1,25(OH)2D3 in a cell type-specific manner in prostate cancer cells.

Parathyroid Hormone-Related Protein Interacts With the Transforming Growth Factor-ß/Bone Morphogenetic Protein-2/Gremlin Signaling Pathway to Regulate Proinflammatory and Profibrotic Mediators in Pancreatic Acinar and Stellate Cells.

OBJECTIVES: Transforming growth factor ß (TGF-ß) regulates immune and fibrotic responses of chronic pancreatitis. The bone morphogenetic protein 2 (BMP-2) antagonist gremlin is regulated by TGF-ß. Parathyroid hormone-related protein (PTHrP) levels are elevated in chronic pancreatitis. Here, we investigated the cross-talk between TGF-ß/BMP-2/gremlin and PTHrP signaling. METHODS: Reverse transcription/real-time polymerase chain reaction, chromatin immunoprecipitation, Western blotting, and transient transfection were used to investigate PTHrP regulation by TGF-ß and BMP-2 and gremlin regulation by PTHrP. The PTHrP antagonist PTHrP (7-34) and acinar cells with conditional Pthrp gene deletion (PTHrP) were used to assess PTHrP's role in the proinflammatory and profibrotic effects of TGF-ß and gremlin. RESULTS: Transforming growth factor ß increased PTHrP levels in acinar cells and pancreatic stellate cells (PSCs) through a Smad3-dependent pathway. Transforming growth factor ß's effects on levels of IL-6 and intercellular adhesion molecule 1 (ICAM-1) (acinar cells) and procollagen I and fibronectin (PSCs) were inhibited by PTHrP (7-34). PTHrP suppressed TGF-ß's effects on IL-6 and ICAM-1. Parathyroid hormone-related hormone increased gremlin in acinar cells, and inhibiting gremlin action suppressed TGF-ß's and PTHrP's effects on IL-6 and ICAM-1. Transforming growth factor ß-mediated gremlin up-regulation was suppressed in PTHrP cells. Bone morphogenetic protein 2 suppressed PTHrP levels in PSCs. CONCLUSIONS: Parathyroid hormone-related hormone functions as a novel mediator of the proinflammatory and profibrotic effects of TGF-ß. Transforming growth factor ß and BMP-2 regulate PTHrP expression, and PTHrP regulates gremlin levels.

PTH-related protein enhances LoVo colon cancer cell proliferation, adhesion, and integrin expression.

Parathyroid hormone-related protein (PTHrP) has been localized in human colon cancer tissue and cell lines. Tumor cell adhesion to extracellular matrix (ECM) proteins plays a major role in the invasion and metastasis of tumor cells, and is mediated via integrin subunits. The LoVo human colon cancer cell line was used as a model system to study the effects of PTHrP on cell proliferation and adhesion to ECM proteins found in normal liver. Clones of LoVo cells engineered to overexpress PTHrP by stable transfection with a PTHrP cDNA showed enhanced cell proliferation vs. control (empty vector-transfected) cells. PTHrP-overexpressing cells also showed significantly higher adhesion to collagen type I, fibronectin, and laminin, and enhanced expression of the [symbol: see text] integrin subunits. These results indicate that PTHrP may play a role in colon cancer invasion and metastasis by increasing cell proliferation and adhesion to the ECM via upregulation of proinvasive integrin expression.

Restoration of the anti-proliferative and anti-migratory effects of 1,25-dihydroxyvitamin D by silibinin in vitamin D-resistant colon cancer cells.

Colorectal carcinoma (CRC) is the third most common cancer in developed countries. A large fraction of cases are linked to chronic intestinal inflammation, with concomitant increased TNF-α release and elevated Snail1/Snail2 levels. These transcription factors in turn suppress vitamin D receptor (VDR) expression, resulting in loss of responsiveness to the protective anti-proliferative and anti-migratory effects of 1,25-dihydroxyvitamin D (1,25D). Experimental and epidemiologic evidence support the use of natural products to target CRC. Here we show that the flavonolignan silibinin reverses the TNF-α-induced upregulation of Snail1 and Snail2 in the 1,25D-resistant human colon carcinoma cells HT-29. These silibinin effects are accompanied by an increase in VDR levels; Snail1 overexpression reverses these silibinin effects. Silibinin also restores promoter activity from a vitamin D-response element (VDRE) reporter construct. While 1,25D had no significant effect on HT-29 and SW480-R cell proliferation and migration, co-treatment with silibinin restored 1,25D responsiveness. In addition, co-treatment with silibinin plus 1,25D decreased proliferation and migration at doses where silibinin alone had no effect. These findings demonstrate that this combination may present a novel approach to target CRC in conditions of chronic colonic inflammation.

Role of Parathyroid Hormone-Related Protein Signaling in Chronic Pancreatitis.

Chronic pancreatitis (CP), a progressive inflammatory disease where acini are destroyed and replaced by fibrous tissue, increases the risk for pancreatic cancer. Risk factors include alcohol, smoking, and obesity. The effects of these risk factors are exacerbated in patients with mutations in genes that predispose to CP. The different environmental and genetic factors produce the same clinical phenotype; once CP develops, disease course is the same regardless of etiology. Critical questions still need to be answered to understand what modifies predisposition to develop CP in persons exposed to risk factors. We postulate that risk factors modulate endogenous pathways, with parathyroid hormone-related protein (PTHrP) signaling being one such pathway. In support, PTHrP levels are elevated in mice treated with alcohol, and in mouse models of cerulein- and pancreatic duct ligation-induced CP. Disrupting the Pthrp gene in acinar cells exerts protective effects (decreased edema, histological damage, amylase and cytokine release, and fibrosis) in these CP models. PTHrP levels are elevated in human CP. Currently, CP care lacks specific pharmacological interventions. Targeting PTHrP signaling may present a novel therapeutic strategy that inhibits pancreatic inflammation and fibrosis, especially since the risk of developing pancreatic cancer is strongly associated with duration of chronic inflammation.

Gremlin is a key pro-fibrogenic factor in chronic pancreatitis.

UNLABELLED: The current study aims to identify the pro-fibrogenic role of Gremlin, an endogenous antagonist of bone morphogenetic proteins (BMPs) in chronic pancreatitis (CP). CP is a highly debilitating disease characterized by progressive pancreatic inflammation and fibrosis that ultimately leads to exocrine and endocrine dysfunction. While transforming growth factor (TGF)-ß is a known key pro-fibrogenic factor in CP, the TGF-ß superfamily member BMPs exert an anti-fibrogenic function in CP as reported by our group recently. To investigate how BMP signaling is regulated in CP by BMP antagonists, the mouse CP model induced by cerulein was used. During CP induction, TGF-ß1 messenger RNA (mRNA) increased 156-fold in 2 weeks, a BMP antagonist Gremlin 1 (Grem1) mRNA levels increased 145-fold at 3 weeks, and increases in Grem1 protein levels correlated with increases in collagen deposition. Increased Grem1 was also observed in human CP pancreata compared to normal. Grem1 knockout in Grem1 (+/-) mice revealed a 33.2 % reduction in pancreatic fibrosis in CP compared to wild-type littermates. In vitro in isolated pancreatic stellate cells, TGF-ß induced Grem1 expression. Addition of the recombinant mouse Grem1 protein blocked BMP2-induced Smad1/5 phosphorylation and abolished BMP2's suppression effects on TGF-ß-induced collagen expression. Evidences presented herein demonstrate that Grem1, induced by TGF-ß, is pro-fibrogenic by antagonizing BMP activity in CP. KEY MESSAGES: • Gremlin is upregulated in human chronic pancreatitis and a mouse CP model in vivo. • Deficiency of Grem1 in mice attenuates pancreatic fibrosis under CP induction in vivo. • TGF-ß induces Gremlin mRNA and protein expression in pancreatic stellate cells in vitro. • Gremlin blocks BMP2 signaling and function in pancreatic stellate cells in vitro. • This study discloses a pro-fibrogenic role of Gremlin by antagonizing BMP activity in chronic pancreatitis.

Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breast cancer cells.

PTHrP is secreted by breast cancer cells in vivo and in vitro. In the breast cancer cell line MCF-7, PTHrP overexpression is associated with increased mitogenesis. We used this cell line to study the mechanism for the proliferative effects of PTHrP. Clonal MCF-7 lines were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization signal (NLS). Mutation of the NLS negated the proliferative effects and nuclear trafficking of PTHrP, indicating that increased mitogenesis is mediated via an intracrine pathway. Cells overexpressing wild-type PTHrP were enriched in G2 + M stage of the cell cycle compared with cells overexpressing NLS-mutated PTHrP, indicating an intracrine role for PTHrP in cell cycle regulation. Wild-type PTHrP also protected MCF-7 cells from serum starvation-induced apoptosis. Cells overexpressing wild-type PTHrP showed significantly greater cell survival than cells overexpressing NLS-mutated PTHrP. The ratios of the apoptosis-regulating proteins Bcl-2 to Bax and Bcl-x(L) to Bax were higher in cells overexpressing wild-type, but not NLS-mutated, PTHrP compared with control cells. These findings suggest that the proliferative effects of PTHrP in breast cancer cells are mediated through regulation of the cell cycle and apoptosis, and that controlling PTHrP production in breast cancer may be therapeutically useful.

PTH-related protein modulates PC-3 prostate cancer cell adhesion and integrin subunit profile.

Parathyroid hormone-related protein (PTHrP), which has been localized in prostate cancer tissue and cell lines, plays a role in the development of bone metastases, a frequent complication in prostate cancer patients. Tumor cell adhesion to extracellular matrix (ECM) components is mediated via integrin subunits, and plays a major role in the invasion and metastasis of tumor cells. The present experiments examined the ability of PTHrP to influence adhesion of the human prostate cancer cell line PC-3 to several ECM proteins found in normal tissues. Clonal PC-3 cells induced to overexpress PTHrP by stable transfection with PTHrP complementary DNA showed significantly higher adhesion to collagen type 1, fibronectin, and laminin than control (empty vector-transfected) cells. PTHrP-overexpressing cells also exhibited higher expression of the alpha1, alpha5, alpha6, and beta4 integrin subunits. These results suggest that PTHrP may play a role in prostate tumor invasion and metastasis by influencing cell adhesion to the ECM via upregulation of specific integrin subunits.

Prostate cancer cell type-specific regulation of the human PTHrP gene via a negative VDRE.

Parathyroid hormone-related protein (PTHrP) is expressed by prostate cancer cells. Since PTHrP increases the growth and enhances the osteolytic effects of prostate cancer cells, it is important to control the level of PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its non-calcemic analogue, EB1089, suppress PTHrP mRNA and protein levels in the human prostate cancer cell lines PC-3 and LNCaP. The human PTHrP gene contains a sequence element homologous to the negative vitamin D response element within the parathyroid hormone gene. This DNA sequence (nVDRE(hPTHrP)) bound the vitamin D receptor (VDR) present in nuclear extracts from both PC-3 and LNCaP cells. However, when cloned upstream of the SV40 promoter and transiently transfected into PC-3 and LNCaP cells, nVDRE(hPTHrP) downregulated promoter activity in response to 1,25(OH)2D3 or EB1089 treatment in LNCaP, but not in PC-3, cells. These results may help to explain why some prostate cancers appear to be refractory to treatment with vitamin D analogues.

Regulation of PTH-related protein gene expression by vitamin D in PC-3 prostate cancer cells.

Parathyroid hormone-related protein (PTHrP) is expressed by prostate cancer cells. Since PTHrP increases prostate cancer cell growth and enhances the osteolytic effects of prostate cancer cells, it is important to control PTHrP expression in prostate cancer. Vitamin D exerts a protective effect against prostate cancer through its antiproliferative actions. We investigated whether this steroid also downregulates PTHrP gene transcription, using the human prostate cancer cell line PC-3 as a model system. We report that PTHrP mRNA and secreted protein levels are downregulated by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) via a transcriptional mechanism. We also show that PTHrP gene expression is upregulated, also via a transcriptional mechanism, by epidermal growth factor (EGF), which is normally secreted by prostate cancer cells. 1,25(OH)(2)D(3) reversed the EGF-induced PTHrP upregulation at both the mRNA and protein levels. Since PTHrP enhances prostate cancer cell growth, this study demonstrates the importance of maintaining adequate levels of 1,25(OH)(2)D(3).

Parathyroid hormone-related protein upregulates integrin expression via an intracrine pathway in PC-3 prostate cancer cells.

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissue and prostate cancer cell lines, and enhances prostate tumor cell growth both in vivo and in vitro. PTHrP expression also plays a role in the development of bone metastasis, which is a frequent complication in patients with prostate carcinoma. Tumor cell adhesion to extracellular matrix (ECM) components is mediated via integrin subunits, and plays a major role in the invasion and metastasis of tumor cells. We previously showed that PTHrP overexpression increases adhesion of the human prostate cancer cell line PC-3 to the ECM molecules collagen type I, fibronectin, and laminin. Increased adhesion is accompanied by upregulation in the expression of alpha1, alpha5, alpha6, and beta4 integrin subunits. We used the same cell line to study the mechanism via which PTHrP upregulates integrin expression. Clonal PC-3 cells were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization sequence (NLS). Mutation of the NLS negated the effects of PTHrP on alpha1, alpha5, alpha6, and beta4 integrin expression, indicating that these effects are mediated via an intracrine pathway requiring nuclear localization. Expression of the alpha2, alpha3, alphav, and beta1 integrin subunits were comparable in wild-type and NLS-mutated PTHrP transfectants. These findings indicate that PTHrP may play a role in prostate tumor invasion and metastasis by upregulating the expression of specific integrin subunits via an intracrine pathway.

Parathyroid hormone-related protein enhances PC-3 prostate cancer cell growth via both autocrine/paracrine and intracrine pathways.

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissue and prostate cancer cell lines, and positively influences primary prostate tumor growth in vivo. The human prostate cancer cell line PC-3, which expresses functional PTH/PTHrP receptors, was used as a model to study the effects of PTHrP on prostate cancer cell growth. Addition of PTHrP (1-34), (1-86), and (1-139) increased cell number and [3H]thymidine incorporation; these effects were reversed by anti-PTHrP antiserum. This antiserum also decreased endogenous PC-3 cell growth. Clonal PTHrP-overexpressing PC-3 cell lines also showed enhanced cell growth and [3H]thymidine incorporation and were enriched in the G2+M phase of the cell cycle, suggesting an effect of PTHrP on mitosis. Overexpression of PTHrP with the nuclear localization sequence (NLS) deletion partially reversed the growth-stimulatory effects. The growth rate of these cells was midway between that of wild-type PTHrP-overexpressing and control cells, presumably because NLS-mutated PTHrP is still secreted and acts through the cell surface PTH/PTHrP receptor. In contrast to NLS-mutated PTHrP, wild-type protein showed preferential nuclear localization. These results suggest that the proliferative effects of PTHrP in PC-3 cells are mediated via both autocrine/paracrine and intracrine pathways, and that controlling PTHrP production in prostate cancer may be therapeutically beneficial.