High Expression of Parathyroid Hormone-related Protein and Tumor Necrosis Factor-α in Cancer Cells as Risk Factors for Hypercalcemia in Bone Metastases Lytic Lesions

BACKGROUND: Cancer mortality is more commonly due to metastases of the tumor to other organs and the complications that accompany it than the tumor growth itself. Until recently, metastasis has been an insurmountable problem. AIM: As the most frequent site of metastases, apart from the lungs and liver, tumor metastases to bone are associated with hypercalcemia which is fatal for the affected patient. METHODS: This study used a case-control study design. The case group consisted of paraffin block samples derived from bone metastatic cancer cell biopsies of patients with hypercalcemic lytic lesions. The control group consisted of paraffin block samples derived from bone metastatic cancer cell biopsies of patients with non-hypercalcemic lytic lesions. Radiological examination was performed to examine the presence of lytic lesions, followed by data collection of serum calcium levels. The data obtained from the histopathological examination was confirmed along with the availability of paraffin blocks of bone metastasis cancer cell biopsy samples, and immunohistochemical analysis was performed to determine the expression of tumor necrosis factor-α _(TNF-α) and parathyroid hormone-related protein (PTHrP). A Mann–Whitney test was performed to determine the expression of TNF-α _and PTHrP between hypercalcemia and non-hypercalcemia groups. To identify the cut-off point, Youden index on receiver operating characteristic was used, then the optimal cut-off point was determined where the sensitivity and specificity curves intersect. Analysis of risk factor assessment was done by creating a 2 × 2 cross-tabulations and calculating the association size in the form of odds ratio (OR). RESULTS: The expression of PTHrP and TNF-α _in the case group was significantly different from the control group with p < 0.05. The cut-off point for PTHrP expression was 267.5 with an area under the curve of 0.93, indicating a high accuracy value. The cut-off point for TNF-α _expression was 227.5 with an area under the curve of 0.68, indicating a moderate accuracy value. The OR between hypercalcemia and non-hypercalcemia to PTHrP expression was 110.3 (Fisher’s exact statistical test obtained p < 0.05), while the OR between hypercalcemia and non-hypercalcemia to TNF-α _expression was 7.27 (Fisher’s exact test statistical obtained p = 0.01). CONCLUSION: Significant differences in the expression of PTHrP and TNF-α _were found between patients with bone metastases lytic lesions with hypercalcemia compared to those without hypercalcemia. We can conclude that either a high level of PTHrP expression and/or TNF-α _expression in cancer cells can serve as risk factors for hypercalcemia in patients with bone metastatic lytic lesions.

Region-specific effects of blocking estrogen receptors on longitudinal bone growth

Estrogen receptors (ERs) regulate the development of the growth plate (GP) by binding to estrogen, a phenomenon that determines the growth of skeletal bone. However, the exact mechanisms underlying the regulatory effects of ERs on axial and appendicular growth plates during puberty remain unclear. In the present study, the strategy of ERβ blocking resulted in increased longitudinal elongation of the appendicular bone (P<0.01), whereas ERα blocking suppressed appendicular elongation (P<0.05). Blocking both ERs did not have opposite effects on axial longitudinal growth. The expression of chondrocyte proliferation genes including collagen II, aggrecan, and Sox9 and hypertrophic marker genes including collagen X, MMP13, and Runx2 was significantly increased in the growth plate of female mice treated with ERβ antagonist compared with that in the GP of control mice (P<0.05). There were no significant differences in local insulin-like growth factor 1 (IGF-1) expression among these groups (P>0.05), and Indian hedgehog protein (Ihh) and parathyroid related protein (PTHrP) expression differed among these groups (P<0.05). ERs appeared not to affect axial bone growth during puberty in female mice (P<0.05). Our data show that the blocking of different ER subtypes might have a region-specific influence on longitudinal appendicular and axial growth.

Regulation of Hedgehog signaling Offers A Novel Perspective for Bone Homeostasis Disorder Treatment

The hedgehog (HH) signaling pathway is central to the regulation of bone development and homeostasis. HH signaling is not only involved in osteoblast differentiation from bone marrow mesenchymal stem cells (BM-MSCs), but also acts upstream within osteoblasts via the OPG/RANK/RANKL axis to control the expression of RANKL. HH signaling has been found to up-regulate parathyroid hormone related protein (PTHrP) expression in osteoblasts, which in turn activates its downstream targets nuclear factor of activated T cells (NFAT) and cAMP responsive element binding protein (CREB), and as a result CREB and NFAT cooperatively increase RANKL expression and osteoclastogenesis. Osteoblasts must remain in balance with osteoclasts in order to avoid excessive bone formation or resorption, thereby maintaining bone homeostasis. This review systemically summarizes the mechanisms whereby HH signaling induces osteoblast development and controls RANKL expression through PTHrP in osteoblasts. Proper targeting of HH signaling may offer a therapeutic option for treating bone homeostasis disorders.

Parathyroid hormone-related protein modulates inflammation in mouse mesangial cells and blunts apoptosis by enhancing COX-2 expression

Injury of mesangial cells (MC) is a prominent feature of glomerulonephritis. Activated MC secrete inflammatory mediators that induce cell apoptosis. Parathyroid hormone-related peptide (PTHrP) is a locally active cytokine that enhances cell survival and is upregulated by proinflammatory factors in many cell types. The aim of this study was to analyze the regulation of PTHrP expression by inflammatory cytokines and to evaluate whether PTHrP itself acts as a proinflammatory and/or survival factor on male murine MC in primary culture. Our results showed that IL-1β (10 ng/ml) and TNF-α (10 ng/ml) rapidly and transiently upregulated PTHrP expression in MC. The effects of IL-1β were both transcriptional and posttranscriptional, with stabilization of the PTHrP mRNA by human antigen R (HuR). Proteome profiler arrays showed that PTHrP itself enhanced cytokines within 2 h in cell lysates, mainly IL-17, IL-16, IL-1α, and IL-6. PTHrP also stimulated sustained expression (2–4 h) of chemokines, mainly regulated upon activation normal T cell expressed and secreted (RANTES)/C-C motif chemokine 5 (CCL5) and macrophage inflammatory protein-2 (MIP-2)/C-X-C motif chemokine 2 (CXCL2), thymus and activation-regulated chemokine (TARC)/CCL17, and interferon-inducible T cell α-chemoattractant (I-TAC)/CXCL11. Moreover, PTHrP markedly enhanced cyclooxygenase-2 (COX-2) expression and elicited its autoinduction through the activation of the NF-κB pathway. PTHrP induced MC survival via the COX-2 products, and PTHrP overexpression in MC blunted the apoptotic effects of IL-1β and TNF-α. Altogether, these findings suggest that PTHrP functions as a booster of glomerular inflammatory processes and may be a negative feedback loop preserving MC survival.

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-β (TGF-β) 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-β, a key effector of pancreatic pathophysiology. This novel network may be amenable to pharmacologic manipulations during CP in humans.

Muenke Syndrome Mutation, FgfR3P244R, Causes TMJ Defects

Muenke syndrome is characterized by various craniofacial deformities and is caused by an autosomal-dominant activating mutation in fibroblast growth factor receptor 3 ( FGFR3P250R). Here, using mice carrying a corresponding mutation ( FgfR3P244R), we determined whether the mutation affects temporomandibular joint (TMJ) development and growth. In situ hybridization showed that FgfR3 was expressed in condylar chondroprogenitors and maturing chondrocytes that also expressed the Indian hedgehog (Ihh) receptor and transcriptional target Patched 1( Ptch1). In FgfR3P244R mutants, the condyles displayed reduced levels of Ihh expression, H4C-positive proliferating chondroprogenitors, and collagen type II- and type X-expressing chondrocytes. Primary bone spongiosa formation was also disturbed and was accompanied by increased osteoclastic activity and reduced trabecular bone formation. Treatment of wild-type condylar explants with recombinant FGF2/FGF9 decreased Ptch1 and PTHrP expression in superficial/polymorphic layers and proliferation in chondroprogenitors. We also observed early degenerative changes of condylar articular cartilage, abnormal development of the articular eminence/glenoid fossa in the TMJ, and fusion of the articular disc. Analysis of our data indicates that the activating FgfR3P244R mutation disturbs TMJ developmental processes, likely by reducing hedgehog signaling and endochondral ossification. We suggest that a balance between FGF and hedgehog signaling pathways is critical for the integrity of TMJ development and for the maintenance of cellular organization.

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.