Comparative tissue distribution of the processing enzymes “prohormone thiol protease”, and prohormone convertases 1 and 2, in human PTHrP-producing cell lines and mammalian neuroendocine tissues

Peptide hormones are generated by proteolytic processing of their respective protein precursors by several prohormone processing proteases. The peptide hormone PTHrP is widely expressed in normal and malignant tissues, where proPTHrP undergoes proteolytic processing to generate PTHrp peptides with distinct biological actions. In this study, the tissue distribution of the prohormone processing enzymes PTP, PC1, and PC2 were compared by immunohistochemistry in human PTHrP-producing cancer cell lines, and in mammalian neuroedocrine and other tissues from rat and bovine that contain peptide hormones. PTP, PC1, and PC2 were prominently expressed in PTHrP-expressing human cancer cell lines originating from tumors of the breast, lung prostate, as well as lymphoma. These processing enzymes also showed significant expression in normal mammalian neuroendocrine tissues from bovine and rat, including pituitary, hypothalamus, adrenal medulla, pancreas, and oter tissue. Most neuroendocrine tissues contained prominent levels of at least two of the three processing enzymes examined, and all tissues contained at least one of these three enzymes. Differential expression of processing enzyme proteins was also demonstrated by Western blots. The differential expression of PTP, PC1 and PC2 observed in certain cancer and normal neuroendocrine cell types postulates selective roles for these processing enzymes in different tissues for generating biologically active peptide hormones. Theses results support the importnce of these processing enzymes in their hypothesized roles in prohormone processing.     

Alteration of secretion of parathyroid hormone-related peptide and expression of its mRNA in a human hepatoma cell line (HEP G2) treated with agents that affect cell growth

Previously, using human hepatoma cells (HepG2), we found that immunoneutralization of secreted PTHrP increased cell growth. Here we asked whether PTHrP production was affected by agents that alter growth of Hep G2 cells. Immunoreactive PTHrP in medium and PTHrP mRNA expression were examined. Treatment of cells with 10 μM hydrocortisone or 1 ng/mL TGF-β1 for 72 h inhibited cell growth by 28±6 and 36±2% and increased PTHrP in medium by 128±10 and 525 ±27%, respectively. The increase in PTHrP produced by both agents was dose-and time-dependent, and the increased PTHrP was accompanied by dose-and time-dependent enhanced expression of PTHrP mRNA. In contrast, 10% fetal bovine serum (FBS) for 72 h increased cell growth by 38±6% (vs serum-free medium) and decreased PTHrP production by 49±4% whereas culture in high glucose (3–4g/L) increased cell growth by 43±1% (vs 1 g/L glucose) and decreased PTHrP by 55±0.4%. Inhibition of PTHrP by both FBS and glucose was dose-dependent; FBS also inhibited PTHrP mRNA. The results show that increased cell growth was associated with decreased PTHrP production, while decreased growth was accompanied by increased PTHrP production. The findings imply that PTHrP may help mediate growth effects of these agents on Hep G2 cells.     

甲状旁腺相关蛋白在鼠胚髁突外植体软骨内成骨中的作用

目的探讨甲状旁腺相关蛋白(PTHrP)对体外培养的鼠胚髁突软骨内成骨的影响。方法体外解剖分离鼠胚髁突,行外植体培养,通过组织学、免疫组化等方法观察PTHrP对体外培养的髁突外植体软骨内成骨的影响。结果髁突外植体在无血清半固态培养系统中能正常发育。加入人PTHrP(1-34)后,实验组髁突长度的增加较对照组明显,两组间差异有显著性(P<0.05);组织学及免疫组化染色显示:加入人PTHrP(1-34)后培养的髁突外植体增殖层和肥大软骨细胞层明显增厚,同时Ⅱ及Ⅹ型胶原在增殖层和肥大软骨细胞层中表达增强。结论PTHrP可刺激髁突外植体软骨增殖层和肥大层软骨细胞的增殖,促进髁突软骨内成骨的形成。?     

甲状旁腺素相关肽治疗绝经后骨质疏松症的研究进展

甲状旁腺素相关肽(parathyroid hormone-related peptide,PTHrP)是人体内存在的一种分泌蛋白,它因与甲状旁素(parathyroid hormone,PTH)在分子结构和信号传导方面有很高的同源性,作为潜在的骨形成促进剂运用于治疗骨质疏松症。PTHrp通过各种信号传导通路调控骨代谢,发挥影响成骨细胞和破骨细胞的作用。并已在动物试验中被证实能有效地促进骨骼合成,改善骨微结构,提高骨量以及增加骨强度。相关药物Abaloparatide的临床研究证实其可显著增加骨密度,改善骨代谢,降低骨折风险,对绝经后骨质疏松有一定的治疗效果。     

Bone metastasis: Interaction between cancer cells and bone microenvironment

BackgroundBone is one of the most common target organs for cancer metastasis, especially in patients with advanced breast and prostate cancers. Despite recent advances in therapeutic approaches, bone metastases remain incurable and produce multiple complications called skeletal-related events, including hypercalcemia, pathological fractures, spinal compression, and bone pain, which are associated with poor prognosis.HighlightAlthough the precise mechanisms are yet to be fully elucidated, accumulating evidence suggests that bone provides a favorable microenvironment that enables circulating cancer cells to home, proliferate, and colonize, resulting in the formation of metastasis. Cancer cells that metastasize to bone also possess unique features, enabling them to utilize the bone microenvironment. Thus, communication between cancer cells and bone is believed to be critical for the development and progression of bone metastases.ConclusionContinued studies are warranted to understand the molecular mechanisms underlying bone metastases and to develop mechanism-based and effective therapeutic interventions.     

p21 and Parathyroid Hormone-Related Peptide in the Growth Plate

It is essential for terminal chondrocytes to die before the conversion of calcified cartilage to bone. We have previously demonstrated that apoptosis occurred in the terminal hypertrophic chondrocyte of the growth plate. However, the essential mechanism by which the differentiation of chondrocytes is regulated has not yet been characterized. The purpose of this study was to investigate the mechanism for regulating chondrocyte differentiation. We focused on PTHrP and p21 which regulated the differentiation of chondrocytes and investigated how these factors interacted with each other in chondrocyte differentiation in the growth plate. PTHrP was strongly positive on immunostaining at the interface between the proliferating and the upper zone of the hypertrophic chondrocytes, whereas p21 was negative. On the other hand, p21 was positive in the lower zone of hypertrophic chondrocytes. Furthermore, PTHrP up-regulated the cell proliferation and down-regulated the expression of the p21 messengers in SW-1353 chondrosarcoma cells. These findings indicated that PTHrP might be a negative regulator for p21 in the differentiation of chondrocytes. Received: 1 March 2000 / Accepted: 25 May 2000 / Online publication: 2 November 2000     

Nuclear targeting of a midregion PTHrP fragment is necessary for stimulating growth in breast cancer cells

Parathyroid-hormone related protein (PTHrP) is the primary factor in humoral hypercalcemia of malignancy and is highly secreted by breast cancers. The pro-hormone undergoes post-translational processing and cleavage to give rise to mature secretory peptides, one of which is midregion PTHrP (38-94/95/101) containing a nuclear localisation sequence (NLS) in amino acids (87-106). The current study investigates whether the NLS in midregion PTHrP is important in breast cancer growth. PTHrP-(67-101), a midregion PTHrP fragment containing NLS-(87-101) significantly increased growth of MCF-7 and MDA-MB231 cells (126.3 and 121.3% of control respectively in serum conditions), independent of PTHR1 whereas PTHrP-(67-86), which lacks the NLS did not. Fluorescent-labelled PTHrP-(67-101) translocated to the nucleus, whereas PTHrP-(67-86) remained cytosolic and a scrambled(+NLS) peptide was not internalised. In comparison, no growth influence or uptake was seen in non-tumour breast cells (Hs578Bst). Increases in intracellular calcium mobilisation were observed in breast cancer cells stimulated with both PTHrP-(67-101) and PTHrP-(67-86) (EC(50) of 3.2 pM and 2.2 pM respectively for MCF-7 cells), whereas inositide turnover was not detected. Both nuclear uptake and calcium signalling were attenuated in the presence of EGTA, but not with U73122 or N-terminal PTHrP peptides. Our studies indicate that the NLS-containing midregion PTHrP peptide is dependent on both internalisation and nuclear translocation to induce growth in breast cancer cells. These findings highlight the importance of midregion PTHrP and its receptor in breast cancer growth and may provide potential targets for future therapeutic intervention.     

Biological action of parathyroid hormone (PTH)-related peptide (PTHrP) mediated either by the PTH/PTHrP receptor or the nucleolar translocation in chondrocytes

Parathyroid hormone (PTH)-related peptide (PTHrP) has been believed to act by binding the common receptor to PTH (PTH/PTHrP receptor). However, PTHrP is localized not only in the secretory pathway, but also in nucleoli by virtue of its nucleolar targeting signal (NTS). This review demonstrates the bipartite action of PTHrP on chondrocytes, the receptor-mediated and -independent signaling pathway. Mice with deletion of the PTHrP gene were characterized by a chondrodysplasia due to markedly reduced proliferation of epiphyseal chondrocytes. The PTH/PTHrP receptor was localized mainly in proliferative chondrocytes in the epiphyseal cartilage, indicating that PTHrP modulates normal proliferation via the receptor. In contrast to the receptor-mediated action, the mid-region of the amino acid sequence of PTHrP contains an NTS. The PTHrP-translation was found to initiate from both methionine-coding AUG and downstream leucine-coding CUGs in its signal sequence. When translated from CUGs, PTHrP accumulated in the nucleoli, and the translation from AUG localized PTHrP in both the Golgi apparatus and nucleoli. Therefore, nucleolar PTHrP appears to be derived from the translation initiating from both AUG and CUGs. A chondrocytic cell line expressing a full-length PTHrP, but not PTHrP lacking NTS, were resistant to apoptosis caused by serum depletion, suggesting that the nucleolar PTHrP in chondrocytes serves as a survival factor against apoptosis. Thus, PTHrP regulates chondrocyte proliferation, differentiation and apoptosis by mediating its receptor or acting directly on the nucleolus.     

间断性PTHrP对成牙骨质细胞凋亡及矿化的影响

目的 探讨间歇性甲状旁腺激素相关蛋白(parathyroid hormone related protein, PTHrP)刺激在成牙骨质细胞中对细胞凋亡和成牙骨质矿化相关蛋白和细胞因子表达的影响。方法 应用成牙骨质细胞OCCM-30,使用PTHrP(1-36)、甲状旁腺激素I型受体(parathyroid hormone type I receptor, PTH1R)阻断剂PTHrP(7-34)对细胞进行间歇性刺激,间歇性给药模式包含3个周期,48 h/周期,分为对照组、PTHrP(1-36)组及 PTH1R阻断剂组。采用流式细胞术检测细胞凋亡;采用茜素红染色观察矿化功能;采用real-time PCR和Western blotting法检测细胞内成牙骨质矿化相关蛋白-骨桥素(osteopontin, OPN)、I型胶原蛋白(collagen-1, COL-1)及成牙骨质相关细胞因子I型胰岛素样生长因子-1(insulin like growth factor-1, IGF-1)的基因表达及蛋白质表达。结果 间断性PTHrP抑制成牙骨质细胞凋亡,PTH1R阻断剂促进成牙骨质细胞凋亡(P<0.05);PTHrP间断性刺激能够显著增加成牙骨质细胞内OPN、COL-1、IGF-1基因及蛋白表达(P<0.05);PTH1R阻断剂抑制成牙骨质细胞内OPN、COL-1、IGF-1基因及蛋白表达(P<0.05)。结论 间断性PTHrP可通过与成牙骨质细胞上PTH1R相互作用抑制细胞凋亡,促进成牙骨质细胞矿化以及相关蛋白和细胞因子的表达。     

PTH-related peptide-like immunoreactivity in the median emminence, paraventricular and supraoptic nuclei in colchicine-treated rats

The existence of PTH-related peptide (PTHrP) in the hypothalamus was examined by immunohistochemistry in colchicine-treated rats. Two days after intracerebroventricular administration of colchicine dense PTHrP-like immunoreactivity (LI) was observed in the external zone of the median emminence (ME). PTHrP-LI cells were found in the paraventricular nucleus, the supraoptic nucleus and the periventricular region of the third ventricule. The effects of PTHrP on intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were examined by a Ca²⁺ imaging method using fura-2 in perifused preparations of isolated rat anterior pituitary cells. The rise in [Ca²⁺]_i induced by PTHrP was found in approximately 17% of the cells examined. These results suggest that PTHrP-LI cells in the hypothalamus may project to the ME and contribute to the anterior pituitary function.