Parathyroid Hormone Related-protein Induction Research Articles

Serotonin stimulated parathyroid hormone related protein induction in the mammary epithelia by transglutaminase-dependent serotonylation.

Mammary-derived serotonin has been implicated in breast-to-bone communication during lactation by increasing parathyroid hormone related-protein (PTHrP) in the mammary gland. It is well established that PTHrP acts on the bone to liberate calcium for milk synthesis during lactation; however, the mechanism of serotonin’s regulation of PTHrP has not been fully elucidated. Recently, serotonylation has been shown to be involved in a variety of physiological processes mediated by serotonin. Therefore, we investigated whether serotonylation is involved in serotonin’s regulation of PTHrP in the mammary gland using lactogenically differentiated mouse mammary epithelial cells. We investigated the effect of increased intracellular serotonin using the antidepressant fluoxetine or 5-hydroxytryptophan (serotonin precursor), with or without transglutaminase inhibition and the corresponding action on PTHrP induction and activity. Treatment with fluoxetine or 5-hydroxytryptophan significantly increased intracellular serotonin concentrations and subsequently increased PTHrP gene expression, which was reduced with transglutaminase inhibition. Furthermore, we determined that transglutaminase activity is increased with lactogenic differentiation and 5-hydroxytryptophan or fluoxetine treatment. We investigated whether RhoA, Rac1, and Rab4 were potential serotonylation target proteins. We speculate that RhoA is potentially a serotonylation target protein. Our data suggest that serotonin regulates PTHrP induction in part through the process of serotonylation under lactogenic conditions in mouse mammary epithelial cells.

Abstract 2042: Modeling estrogen receptor-positive breast cancer bone metastasis to query osteolytic effects of tumor ER signaling

Abstract Estrogen receptor α-positive (ER+) breast cancers (BrCAs) have the greatest predilection for forming clinically evident bone metastases (BMETs). To query ERα’s role in osteolytic BrCA BMET progression, tumoral vs bone microenvironment effects of 17β-estradiol (E2) were determined in a human xenograft murine model of ER+ BrCA BMET. Female athymic mice aged 4 weeks (young) or 15 weeks (mature) were inoculated with 1x105 human ER+ MCF-7 BrCA cells via the left cardiac ventricle 2 days post-placement of 60-day release E2 pellets (0.05, 0.10, 0.18, 0.36, & 0.72 mg E2). Osteolytic BMET formation was assessed radiographically and E2effects on bone were determined by DXA and microCT at various time points. The BMET size and proliferation were assessed by immunohistochemical (IHC) analyses at the end of the experiment (42 days). The effect of E2 on tumoral secretion of the osteolytic factor, parathyroid hormone related protein (PTHrP), was determined using a commercially available radioimmunoassay. The incidence and size of osteolytic BMET, which were not evident in the absence of E2 supplementation, were E2 dose-dependent in young mice. In contrast, E2 effects on the bone microenvironment were not dose-dependent, and resulted in identical increases in bone mineral density (BMD) and bone volume (BV/TV). In skeletally immature (young) mice vs skeletally mature mice treated with an identical E2 dose (0.72 mg), which caused significantly different effects on bone turnover, progression of E2-dependent BMET was identical. These results suggest that E2 effects on the tumor, rather than bone, were driving E2-dependency of BMET progression. IHC analysis demonstrated that neither the size of human cytokeratin-positive tumors nor the proportion of Ki67-positive proliferating tumor cells in bone were E2 dose-dependent, suggesting that proliferative effects of E2 could not explain E2-dose dependent differences in osteolytic BMET formation. PTHrP, an osteolytic factor expressed in most clinical BrCA BMET, was E2-inducible ex vivo and secreted in higher levels by tumor cells isolated from BMETs. These results suggest that during ER+ BrCA BMET progression, tumoral effects of E2 not only support ER+ BMET proliferation, but may also have bone-specific effects due to the induction of PTHrP, which may explain the marked osteolytic capacity of the ER+ tumor cells in this model despite a microenvironment where bone volume is markedly increased. Citation Format: Julia N. Cheng, Jennifer B. Frye, Susan A. Whitman, Andrew G. Kunihiro, Madison M. Egan, Julia A. Brickey, Lily A. Alvord, Janet L. Funk. Modeling estrogen receptor-positive breast cancer bone metastasis to query osteolytic effects of tumor ER signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2042.

Loss of RUNX3 expression inhibits bone invasion of oral squamous cell carcinoma.

High recurrence and lower survival rates in patients with oral squamous cell carcinoma (OSCC) are associated with its bone invasion. We identified the oncogenic role of RUNX3 during bone invasion by OSCC. Tumor growth and the generation of osteolytic lesions were significantly inhibited in mice that were subcutaneously inoculated with RUNX3-knockdown human OSCC cells. RUNX3 knockdown enhanced TGF-β-induced growth arrest and inhibited OSCC cell migration and invasion in the absence or presence of transforming growth factor-β (TGF-β), a major growth factor abundant in the bone microenvironment. RUNX3 knockdown induced cell cycle arrest at the G1 and G2 phases and promoted G2 arrest by TGF-β in Ca9.22 OSCC cells. RUNX3 knockdown also inhibited both the basal and TGF-β-induced epithelial-to-mesenchymal transition by increasing E-cadherin expression and suppressing the nuclear translocation of β-catenin. In addition, the expression and TGF-β-mediated induction of parathyroid hormone-related protein (PTHrP), one of key osteolytic factors, was blocked in RUNX3-knockdown OSCC cells. Furthermore, treating human osteoblastic cells with conditioned medium derived from RUNX3-knockdown OSCC cells reduced the receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin ratio compared with treatment with conditioned medium from RUNX3-expressing cells. These findings indicate that RUNX3 expression in OSCC cells contributes to their bone invasion and the resulting osteolysis by inducing their malignant behaviors and production of osteolytic factors. RUNX3 alone or in combination with TGF-β and PTHrP may be a useful predictive biomarker and therapeutic target for bone invasion by oral cancer.

TrxR2 deficiencies promote chondrogenic differentiation and induce apoptosis of chondrocytes through mitochondrial reactive oxygen species

Thioredoxin reductase 2 (TrxR2) is a selenium (Se) containing protein. Se deficiency is associated with an endemic osteoarthropathy characterized by impaired cartilage formation. It is unclear whether TrxR2 have roles in cartilage function. We examined the effects of TrxR2 on chondrogenic ATDC5 cells through shRNA-mediated gene silencing of TrxR2. We demonstrated TrxR2 deficiencies could enhance chondrogenic differentiation and apoptosis of ATDC5 cells. TrxR2 deficiencies increased accumulation of cartilage glycosaminoglycans (GAGs) and mineralization. TrxR2 deficiencies also stimulated expression of extracellular (ECM) gene including Collagen II and Aggrecan. The enhanced chondrogenic properties were further confirmed by activation of Akt signaling which are required for chondrogenesis. In addition, TrxR2 deficiencies promoted chondrocyte proliferation through acceleration of cell cycle progression by increase in both S and G2/M phase cell distribution accompanied with induction of parathyroid hormone-related protein (PTHrP). Moreover, TrxR2 deficiencies induced chondrocyte death via apoptosis and increased cell sensitivity to exogenous oxidative stress. Furthermore, TrxR2 deficiencies induced emission of mitochondrial reactive oxygen species (ROS) without alteration of mitochondrial membrane potential and intracellular ATP content. Finally, treatment of TrxR2 deficiency cells with N-acetylcysteine (NAC) inhibited mitochondrial ROS production and chondrocyte apoptosis. NAC also prevented chondrogenic differentiation of TrxR2 deficiency cells by suppression of ECM gene expression, GAGs accumulation and mineralization, as well as attenuation of Akt signaling. Thus, TrxR2-mediated mitochondrial integrity is indispensable for chondrogenic differentiation of ATDC5 cells. TrxR2 deficiency-induced impaired proliferation and death of chondrocytes may be the pathological mechanism of the osteoarthropathy due to Se deficiency. Notably, this study also uncover the roles of mitochondrial ROS which could stimulate cartilage ECM synthesis that offer novel insights for development of therapeutic agent to prevent cartilage degeneration in human disease.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

The cooperation of CREB and NFAT is required for PTHrP-induced RANKL expression in mouse osteoblastic cells.

Parathyroid hormone-related protein (PTHrP) is known to induce the expression of receptor activator of NF-κB ligand (RANKL) in stromal cells/osteoblasts. However, the signaling pathways involved remain controversial. In the present study, we investigated the role of cAMP/protein kinase A (PKA) and calcineurin/NFAT pathways in PTHrP-induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. PTHrP-mediated induction of RANKL expression was significantly inhibited by H89 and FK506, an inhibitor of PKA and calcineurin, respectively. PTHrP upregulated CREB phosphorylation and the transcriptional activity of NFAT. Knockdown of CREB or NFATc1 blocked PTHrP-induced RANKL expression. PTHrP increased the activity of the RANKL promoter reporter that contains approximately 2 kb mouse RANKL promoter DNA sequences. Insertions of mutations in CRE-like element or in NFAT-binding element abrogated PTHrP-induced RANKL promoter activity. Chromatin immunoprecipitation assays showed that PTHrP increased the binding of CREB and NFATc1/NFATc3 to their cognate binding elements in the RANKL promoter. Inhibition of cAMP/PKA and its downstream ERK activity suppressed PTHrP-induced expression and transcriptional activity of NFATc1. CREB knockdown prevented PTHrP induction of NFATc1 expression. Furthermore, NFATc1 and CREB were co-immunoprecipitated. Mutations in CRE-like element completely blocked NFATc1-induced transactivation of the RANKL promoter reporter; however, mutations in NFAT-binding element partially suppressed CREB-induced RANKL promoter activity. Overexpression of CREB increased NFATc1 binding to the RANKL promoter and vice versa. These results suggest that PTHrP-induced RANKL expression depends on the activation of both cAMP/PKA and calcineurin/NFAT pathways, and subsequently, CREB and NFAT cooperate to transactivate the mouse RANKL gene.

Mammary gland serotonin regulates parathyroid hormone-related protein and other bone-related signals

Breast cells drive bone demineralization during lactation and metastatic cancers. A shared mechanism among these physiological and pathological states is endocrine secretion of parathyroid hormone-related protein (PTHrP), which acts through osteoblasts to stimulate osteoclastic bone demineralization. The regulation of PTHrP has not been accounted for fully by any conventional mammotropic stimuli or tumor growth factors. Serotonin (5-HT) synthesis within breast epithelial cells is induced during lactation and in advancing breast cancer. Here we report that serotonin deficiency (knockout of tryptophan hydroxylase-1) results in a reduction of mammary PTHrP expression during lactation, which is rescued by restoring 5-HT synthesis. 5-HT induced PTHrP expression in lactogen-primed mammary epithelial cells from either mouse or cow. In human breast cancer cells 5-HT induced both PTHrP and the metastasis-associated transcription factor Runx2/Cbfa1. Based on receptor expression and pharmacological evidence, the 5-HT2 receptor type was implicated as being critical for induction of PTHrP and Runx2. These results connect 5-HT synthesis to the induction of bone-regulating factors in the normal mammary gland and in breast cancer cells.

Role of the Renin-Angiotensin System on the Parathyroid Hormone–Related Protein Overexpression Induced by Nephrotoxic Acute Renal Failure in the Rat

Parathyroid hormone-related protein (PTHrP), a mitogenic factor for renal cells, is overexpressed in acute renal failure (ARF). Recent data support an association between PTHrP and the renin-angiotensin system in the damaged kidney. The effects of angiotensin II (Ang II) inhibitors (quinapril, enalapril, and/or losartan) on PTHrP and the PTH1 receptor (PTH1R) expression in rats with either folic acid (FA)- or gentamicin-induced ARF were analyzed. The decreased renal function and the PTHrP upregulation and PTH1R downregulation induced by the nephrotoxins were inhibited by the Ang II blockers. In tubuloepithelial cells NRK-52E, the rapid (10 min) increase in PTHrP mRNA by FA, associated with a perinuclear relocalization of Ang II/AT1 receptor, was inhibited by losartan but not candesartan, which traps Ang II receptors at the cell surface. Maximal PTHrP protein overexpression by FA (at 24 to 72 h)-or by exogenous Ang II-was abolished by both Ang II antagonists. PTHrP upregulation by FA was preceded by increased extracellular signal-regulated kinase (ERK) phosphorylation and inhibited by the ERK inhibitor PD098059. FA also activated cAMP response element-binding (CREB) protein, and this was prevented by losartan in these cells. Moreover, PTHrP mRNA overexpression by either FA or Ang II occurred in NRK 52E that were transfected with a CREB construct but not the dominant-negative CREB133 construct. These findings demonstrate that the decreased renal function and PTHrP overexpression in nephrotoxin-damaged kidney depends on renin-angiotensin system. In this setting, intracellular Ang II/AT1 receptor recycling seems to be related to PTHrP induction through ERK and CREB activation in tubuloepithelial cells.

Cyclic Mechanical Strain Regulates the PTHrP Expression in Cultured Chondrocytes via Activation of the Ca2+ Channel

The association between mechanical stimulation and chondrocyte homeostasis has been reported. However, the participation of PTHrP (parathyroid-hormone-related protein) in the mechano-regulation of chondrocyte metabolism remains unclear. We determined whether mechanical stimulation of chondrocytes induces the expression of PTHrP and, further, whether the mechano-modulation of PTHrP is dependent on the maturational status of chondrocytes. Cyclic mechanical strain was applied to rat growth plate chondrocytes at the proliferating, matrix-forming, and hypertrophic stages at 30 cycles/min. Cyclic mechanical strain significantly increased PTHrP mRNA levels in chondrocytes at the proliferating and matrix-forming stages only. The induction of PTHrP was dependent on loading magnitude at the proliferating stage. Using specific ion channel blockers, we determined that mechano-induction of PTHrP was inhibited by nifedipine, a Ca2+ channel blocker. These results suggest that mechanical induction of PTHrP possibly provides the environment for greater chondrocyte replication and matrix formation that would subsequently affect cartilage formation.

Possible role of parathyroid hormone-related protein as a proinflammatory cytokine in atherosclerosis.

Parathyroid hormone-related protein (PTHrP) is a vasodilator peptide. In addition, PTHrP appears to affect vascular growth and to be a mediator of inflammation in rheumatic and brain disorders. We examined the possible role of PTHrP in the inflammatory process in atherosclerosis We immunohistochemically analyzed the cellular localization of PTHrP, the type 1 PTH/PTHrP receptor (PTH1R), and monocyte chemoattractant protein-1 (MCP-1) in 26 human carotid atherosclerotic plaques. The inflammatory region of plaques was characterized by high PTHrP, PTH1R, and MCP-1 immunostaining in relation to the cap (0.75+/-0.1 versus 0.29+/-0.04, 0.5+/-0.1 versus 0.25+/-0.05, 0.72+/-0.2 versus 0.29+/-0.05, respectively; P<0.05). PTHrP and MCP-1 were colocalized in both resident and inflammatory cells in the plaque. Moreover, in cultured vascular smooth muscle cells (VSMC), PTHrP(1-36) increased MCP-1 mRNA (3-fold at 6 hours) and MCP-1 protein (2.5-fold at 24 hours). This effect was inhibited by either PTHrP(7-34) or various protein kinase A inhibitors and by the nuclear factor-kappaB (NF-kappaB) inhibitor parthenolide. Furthermore, PTHrP(1-36) elicited an increase in NF-kappaB activation in VSMC. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin inhibited the PTHrP(1-36) induction of both NF-kappaB activity and MCP-1 overexpression, and this was reversed by mevalonate. PTHrP appears to be a novel proinflammatory mediator in the atheroma lesion and may contribute to the instability of carotid atherosclerotic plaques. Our data provide a new rationale to understand the mechanisms involved in the beneficial effects of statins in atherosclerosis.

Parathyroid hormone-related protein induction in focal stroke: a neuroprotective vascular peptide.

Parathyroid hormone-related protein (PTHrP) is a multifunctional peptide that enhances blood flow in non-central nervous system (CNS) vascular beds by causing vasodilation. PTHrP expression is induced in non-CNS organs in response to ischemia. Experiments were therefore undertaken to determine whether PTHrP can be induced in brain in response to ischemic injury and whether PTHrP can act locally as a vasodilator in the cerebral vasculature, an effect that could be neuroprotective in the setting of stroke. PTHrP expression was examined by Northern analysis and immunohistochemical staining in male Sprague-Dawley rats subjected to permanent middle cerebral artery occlusion (MCAO). Vasodilatory effects of superfused PTHrP(1-34) on pial arterioles were determined by intravital fluorescence microscopy. Effects of PTHrP(1-34) peptide administration on MCAO infarction size reduction were assessed. PTHrP expression was induced in the ischemic hemisphere as early as 4 h after MCAO and remained elevated for up to 24 h. Increased immunoreactive PTHrP at sites of ischemic tissue injury was located in the cerebral microvessels. Superfusion with PTHrP(1-34) peptide for up to 25 min increased pial arteriolar diameter by 30% in normal animals. In animals with permanent MCAO, PTHrP(1-34) peptide treatment significantly decreased cortical infarct size (-47%). In summary, PTHrP expression increases at sites of ischemic brain injury in the cerebrovasculature. This local increase in PTHrP could be an adaptive response that enhances blood flow to the ischemic brain, thus limiting cell injury.

Parathyroid hormone-related protein (PTHrP) induction in reactive astrocytes following brain injury: a possible mediator of CNS inflammation

PTHrP, a peptide induced in parenchymal organs during endotoxemia and in the synovium in rheumatoid arthritis, has recently been shown to be expressed in immature or transformed human astrocytes, but not in normal cells. This finding has led us to postulate that PTHrP might also be induced in reactive astrocytes in inflamed brain and, thus, act as a mediator of CNS inflammation. To test this hypothesis, PTHrP expression was examined following cortical stab wound injury in rats, a classical model of reactive gliosis. To determine whether PTHrP was induced in glia by TNF-α, a known mediator of inflammation in brain and of PTHrP induction in peripheral tissues, and to determine whether PTHrP, in turn, mediated inflammatory changes in glia, in vitro studies with rat astrocytes and glial-enriched mixed brain cells were also undertaken. Consistent with previous reports of PTHrP expression in normal brain, neurons were the primary site of immunoreactive PTHrP expression in the injured cortex 1 day after stab wound injury. Over the subsequent 3 days, specific immunostaining for PTHrP and for GFAP, a marker of reactive astrocytes, appeared in reactive astrocytes at the wound edge and in perivascular astrocytes, reaching a maximum level of expression at the last time point examined (day 4). TNF-α induced PTHrP expression in astrocyte and glial-enriched brain cells in vitro, suggesting that this pro-inflammatory peptide was a possible mediator of PTHrP expression in CNS inflammation. PTHrP(1–34) acted in an additive fashion with TNF-α to induced astrocyte expression of IL-6, a cytokine with demonstrated neuroprotective effects. Astrocyte proliferation was inhibited by PTHrP(1–34) and PTHrP(1–141), acting via a PTH/PTHrP receptor cAMP signaling pathway. These studies suggest that PTHrP, analogous to its regulatory functions in other non-CNS models of inflammation, may be an important mediator of the inflammatory response in brain.

Effect of parathyroid hormone related protein, and dihydrotestosterone on proliferation and ornithine decarboxylase mRNA in human prostate cancer cell lines.

Parathyroid hormone related protein (PTHrP) has been identified as the major hormone responsible for the syndrome of humoral hypercalcemia of malignancy (HHM). Recent studies have shown that a large number of prostate tumors demonstrate the presence of PTHrP despite the fact that prostate cancer is rarely associated with the HHM syndrome. Other studies have indicated that PTHrP behaves as an early response gene, which stimulates ornithine decarboxylase (ODC) enzyme activity, an enzyme, involved in the biosynthesis of polyamines. It is therefore possible that PTHrP regulates prostate tumor cell proliferation via ODC gene expression. In the present study, we evaluated the effects of PTHrP and/or dihydrotestosterone (DHT) treatment on DNA synthesis by thymidine incorporation in androgen-dependent (LnCaP) and androgen-independent (PC3) human prostate adenocarcinoma cell lines. In addition, we utilized Northern blot analysis to investigate the effect of PTHrP [1-34] alone or in combination with DHT on ODC mRNA. PTHrP [1-34] treatment resulted in an increase in thymidine uptake in PC3 cells by 50%, whereas no such increase was seen in LnCaP cells. However, in the LnCaP cells, in the presence of DHT, PTHrP stimulated DNA synthesis to a level greater than that seen with DHT alone. DHT (10 nM) treatment resulted in an induction of PTHrP as well as ODC mRNAs in the androgen-dependent (LnCaP) but not in androgen-independent (PC3) cell line. PTHrP [1-34] treatment resulted in induction of ODC mRNA in the LnCaP cells. Addition of DHT resulted in a further increase in the ODC mRNA expression. These data suggest that PTHrP may play a role in prostate cancer cell proliferation and the increased ODC gene expression may be one possible mechanisms responsible for this phenomenon.

Molecular mechanisms of osteolytic bone metastases.

Breast carcinoma commonly metastasizes to the skeleton in patients with advanced disease to cause bone destruction and the associated pain, hypercalcemia, fracture, and nerve-compression syndromes. In this scenario, the bone destruction is mediated by the osteoclast. Tumor-produced parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption, is a major mediator of the osteolytic process. Transforming growth factor beta (TGFbeta), which is abundant in bone matrix and is released as a consequence of osteoclastic bone resorption, may promote breast carcinoma osteolysis by stimulating PTHrP production by tumor cells. Stable breast carcinoma MDA-MB-231 cell lines were constructed that expressed mutant TGFbeta receptors, Smad proteins, or estrogen receptor (ER)-alpha and were used to determine the role of TGFbeta in modulating tumor production of PTHrP. These stable cell lines were applied to a mouse model of human breast carcinoma metastases to the bone to dissect the molecular mechanisms responsible for osteolytic bone metastases. TGFbeta promoted the development and progression of osteolytic bone metastases by inducing tumor production of PTHrP, the effect of which was mediated through the Smad signaling pathway. PTHrP stimulated osteoclastic bone resorption by increasing osteoblast production of the receptor activator of nuclear factor K B (RANK) ligand and decreasing osteoblast production of osteoprotegerin (OPG). A constitutively active ER-alpha mutation (Tyr537Asn), identified from a human bone metastases, when it was expressed in human breast carcinoma cells, caused increased production of PTHrP. TGFbeta significantly enhanced the ER-alpha-mediated transcriptional activity induced by ER-alpha (Tyr537Asn), and this resulted in further stimulation of PTHrP production. These data indicate a central role for TGFbeta in the pathogenesis of osteolytic bone metastases from breast carcinoma by 1) the induction of PTHrP through the Smad signaling pathway and 2) the potentiation of ER-alpha-mediated transcription induced by a constitutively active ER-alpha. Understanding the mechanisms of osteolysis at a molecular level will generate more effective therapeutic agents for patients with this devastating complication of cancer.

Synergistic induction of parathyroid hormone-related protein in amnion derived (WISH) cells is caused by interleuiqn-1? and tumour necrosis factor-? with transforming growth factor ?

Synergistic induction of parathyroid hormone-related protein in amnion derived (WISH) cells is caused by interleuiqn-1? and tumour necrosis factor-? with transforming growth factor ?