Relaxation of renal arterioles by parathyroid hormone and parathyroid hormone-related protein

The effects of parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) on renal arteriolar tone and proximal tubule adenylate cyclase were examined. In both afferent and efferent arterioles, PTH produced concentration-dependent relaxation of norepinephrine-induced tone with EC50 values of 8.7 and 9.9 nmol/l, respectively. PTHrP also produced relaxations that were indistinguishable from PTH. In proximal convoluted tubules PTH and PTHrP stimulated adenylate cyclase to the same extent and with similar potencies. The PTH antagonist, bPTH(7-34), totally blocked PTH-induced arteriolar relaxation but had no effect on proximal tubule adenylate cyclase. The results demonstrate that PTH and PTHrP are potent relaxants of glomerular arterioles and that PTH receptors present on the renal microvasculature may differ from those present on proximal tubules.     

A role for parathyroid hormone-related protein in the pathogenesis of inflammatory/autoimmune diseases.

Our increased understanding of the critical role of cytokines in chronic inflammatory/autoimmune diseases has led to the recent development of effective anti-cytokine treatments. In particular, agents blocking the function of TNF-alpha, a cytokine first identified as an endotoxin-inducible mediator of tumor cell necrosis, are now licensed for the treatment of rheumatoid arthritis (RA) and inflammatory bowel disease. However, TNF-alpha is but one member of a cytokine network that is responsible for mediating these inflammatory disorders. Therefore, as our understanding of the pathophysiologic role of other members of this inflammatory network increases, other cytokines may similarly be identified as effective targets for treatment. In this article, we will review evidence which suggests that parathyroid hormone-related protein (PTHrP), a peptide which, like TNF-alpha, was first identified because of its effects in the setting of malignancy, may in fact serve an important non-neoplastic, physiologic function by mediating the inflammatory/autoimmune host response. Data identifying PTHrP as a member of the cytokine network induced in multi-organ inflammation and rheumatoid arthritis will be summarized, initial evidence comparing the therapeutic efficacy of PTHrP- vs. TNF-alpha-blockade in the treatment of endotoxemia will be reviewed, and potential future areas of research, including assessment of the effects of PTHrP blockade in the treatment of RA, will be discussed.     

甲状旁腺激素相关蛋白对人间充质干细胞成脂肪分化的影响

目的:观察甲状旁腺激素相关蛋白(PTHrP)在人间充质干细胞体外成脂肪分化中的影响,探讨其作用机制。方法:体外分离培养人间充质干细胞,分为对照组、成脂诱导组和成脂+PTHrP组。采用MTT法测定细胞增殖情况;苏丹红染色法观察成脂肪分化;RT-PCR法检测脂蛋白脂肪酶(LPL)的表达水平。结果:成脂+PTHrP组与成脂诱导组细胞增殖均显著高于对照组(P<0.05);成脂+PTHrP组与成脂诱导组比较,前期无显著性差异,从第4日起有显著性差异(P<0.05)。苏丹红IV染色结果显示,成脂诱导组出现阳性细胞的时间最早,阳性率和强度最强;其LPL mRNA表达水平也显著高于对照组和成脂+PTHrP组(P<0.05)。结论:PTHrP可抑制人间充质干细胞向成脂肪方向的分化。     

甲状旁腺素和甲状旁腺素相关肽在皮肤病中的研究进展

甲状旁腺素（ PTH）和甲状旁腺素相关肽（ PTHrP）是一类多肽类激素,它们具有相似的基因结构、相同的膜受体,在人体钙、磷代谢过程中起着重要的调节作用。 PTH和PTHrP及其受体除表达于肿瘤组织外,在皮肤、毛囊等正常组织也有表达。它们对表皮增殖分化、毛发生长的生理作用等方面,有望成为银屑病等常见皮肤病的治疗新靶点。     

Rat model of the hypercalcaemia induced by parathyroid hormone-related protein: characteristics of three bisphosphonates

In our preliminary experiment, we found that a constant infusion of a high dose of parathyroid hormone-related protein induced both hyperphosphataemia and hypocalcaemia, secondary to renal dysfunction. Therefore, in this study, we developed two types of parathyroid hormone-related protein-induced hypercalcaemia models. One is the hypercalcaemia model, which did not show renal-dysfunction-induced hypocalcaemia. This model might be suitable for estimating hypocalcaemic activities of drugs, especially of those that act on bone resorption. The other is the model for estimating histological changes, which is associated with renal dysfunction. We then used these models to investigate the effects of three different bisphosphonates. Since the hypercalcaemic effect of parathyroid hormone-related protein infusion plateaued at 20 pmol/h, and higher doses of parathyroid hormone-related protein caused an elevation of blood urea nitrogen, the parathyroid hormone-related protein infusion rate was fixed at 20 pmol/h to avoid renal dysfunction and at 40 pmol/h to elicit renal dysfunction. The hypocalcaemic efficiencies of clodronate and etidronate were almost the same but pamidronate was 17.9 times more potent than clodronate. Additionally, both clodronate and pamidronate decreased the plasma concentrations of blood urea nitrogen and the Ca2+ times inorganic P product, whereas etidronate lacked these effects. Clodronate suppressed renal calcification and tubular dilatation in the renal-dysfunction model. These data indicated that clodronate and pamidronate not only decrease the plasma Ca2+ concentration but also improve the renal dysfunction induced by hypercalcaemia.     

Parathyroid hormone and parathyroid hormone-related protein: model systems for the development of an osteoporosis therapy

The parathyroid hormone (PTH) plays a vital role in the homeostasis of calcium within the blood stream. Given its unique ability to increase bone density, an understanding of the molecular mechanism by which the hormone is recognized and binds to its receptor should provide targets for the development of PTH-based, anabolic agents for the treatment of osteoporosis. Parathyroid hormone related protein (PTHrP), a genetically and structurally distinct hormone which displays similar binding and activation profiles as PTH, has greatly facilitated the effort to establish a structure-biological function relationship by allowing for direct comparisons. In an analogous manner, the presence of two receptors, PTH/PTHrP (PTH1) and PTH2, which differ in their ligand selectivity (PTH2 is activated by PTH, not PTHrP) has provided a unique vehicle for probing the structural motifs of the receptor required for ligand recognition and binding. Recent photo-affinity cross-linking studies of PTH and PTHrP binding to PTH1 have produced direct points of contact between the ligand and receptor. Here, we review each of the components involved in this important hormone system, with particular emphasis on the structural features of each: the ligands (PTH and PTHrP), the receptors (PTH1 and PTH2), and the interaction between ligand and receptor. Although the current understanding of the molecular mechanism of ligand binding and receptor activation does not allow for the rational design of drug candidates, and indeed contains much conjecture, significant strides have been made towards this end.     

Effect of intrarenally infused parathyroid hormone-related protein on renal blood flow and glomerular filtration rate in the anaesthetized rat.

1. Parathyroid hormone-related protein (PTHrP) is expressed in the kidney and acts on vascular PTH/ PTHrP receptors to vasodilate the isolated kidney and to stimulate renin release. However, effects of PTHrP on renal blood flow (RBF) and glomerular filtration rate (GFR) in vivo have not been assessed in the absence of its cardiac, peripheral and central effects. We investigated the renal effects of PTH and PTHrP infused into the left renal artery of anaesthetized rats. 2. Intrarenal infusions, adjusted to generate increasing concentrations of human PTHrP(1-34) and rat PTH(1-34) in renal plasma (2 x 10(-11) to 6 x 10(-9) M) produced a comparable dose-dependent increase in RBF. The rise was 4% at the lowest and 34% at the highest concentrations of peptides. Up to a concentration of 2 x 10(-9) M, mean arterial pressure (MAP) and heart rate were not affected, but at 6 x 10(-9) M, intrarenally infused peptides reached the peripheral circulation, and caused a fall in MAP within a few minutes. While MAP returned to basal value after the last peptide infusion, RBF remained more than 10% above control for at least 30 min. 3. Two competitive PTH/PTHrP receptor antagonists, [Nle8,18, Tyr34]-bPTH(3-34)amide and [Leu11, D-Trp12]-hPTHrP(7-34)amide (2 x 10(-8) M) were devoid of agonist activity, but markedly antagonized the dose-dependent increase in RBF elicited by PTHrP. 4. GFR and urine flow were measured in left PTHrP-infused experimental kidney and right control kidney. Renal PTHrP concentration of 10(-10) M elevated left RBF by 10%, and GFR by 20% without significantly increasing filtration fraction, and increased urine flow by 57%. In the right control kidney GFR and diuresis did not change. 5. The results indicate that PTHrP has similar renal haemodynamic effects as PTH and increases RBF, GFR and diuresis in anaesthetized rats.     

Age-related decrease in the effect of parathyroid hormone-related protein on cytosolic free calcium level and tension in rat aortic smooth muscle

Age-related changes in the effect of parathyroid hormone-related protein (PTHrP) on the cytosolic free calcium level ([Ca⁺]_i) and on the tension in rat aortic smooth muscle were investigated. The possible involvement of cAMP, a second messenger of PTHrP, in such changes was also investigated. Spiral aortic strip preparations without endothelium from 8-weeks, 6-months, and 24-months old rats were treated with fura 2/AM, and the fluorescence ratio R340/380, an index of [Ca⁺]_i was measured. Simultaneously, the tension of the preparations was measured. PTHrP-(1–34) and dibutyryl cAMP produced concentration-dependent decreases in the tension and in R340/380 in aortas precontracted with phenylephrine (10^–7 M). These effects were significantly lower in the aortas of 24-months old rats than in the vessels of 8-weeks and 6-months old rats. The effects were similar in the aortas of 8-weeks and 6-months old rats. PTHrP-(1–34) concentration-dependently increased cAMP production in vascular smooth muscle cells (VSMCs) from 8-weeks old rats. However, the increase in cAMP production was significantly lower in cultured VSMCs from 6-months and 24-months old rats than in cells from 8-weeks old rats.These results suggest that the reduced cAMP production stimulated by PTHrP and the reduced effects of cAMP with aging might contribute to the age-related changes in the decreases in tension and [Ca⁺]_i in response to PTHrP in the rat aorta.     

The C-terminal fragment of parathyroid hormone-related peptide promotes bone formation in diabetic mice with low-turnover osteopaenia

BACKGROUND AND PURPOSE

Current data suggest that parathyroid hormone (PTH)-related peptide (PTHrP) domains other than the N-terminal PTH-like domain contribute to its role as an endogenous bone anabolic factor. PTHrP-107-139 inhibits bone resorption, a fact which has precluded an unequivocal demonstration of its possible anabolic action in vivo. We thus sought to characterize the osteogenic effects of this peptide using a mouse model of diabetic low-turnover osteopaenia.

EXPERIMENTAL APPROACH

PTHrP-107-139 was administered to streptozotocin-induced diabetic mice, with or without bone marrow ablation, for 13 days. Osteopaenia was confirmed by dual-energy X-ray absorptiometry and microcomputed tomography analysis. Histological analysis was performed on paraffin-embedded bone tissue sections by haematoxylin/eosin and Masson''s staining, and tartrate-resistent acid phosphatase immunohistochemistry. Mouse bone marrow stromal cells and osteoblastic MC3T3-E1 cells were cultured in normal and/or high glucose (HG) medium. Osteogenic and adipogenic markers were assessed by real-time PCR, and PTHrP and the PTH₁ receptor protein expression by Western blot analysis.

KEY RESULTS

PTHrP-107-139 reversed the alterations in bone structure and osteoblast function, and also promoted bone healing after marrow ablation without affecting the number of osteoclast-like cells in diabetic mice. This peptide also reversed the high-glucose-induced changes in osteogenic differentiation in both bone marrow stromal cells and the more differentiated MC3T3-E1 cells.

CONCLUSIONS AND IMPLICATIONS

These findings demonstrate that PTHrP-107-139 promotes bone formation in diabetic mice. This mouse model and in vitro cell cultures allowed us to identify various anabolic effects of this peptide in this scenario.     

Conformational studies of a potent Leu11, D-Trp12-containing lactam-bridged parathyroid hormone-related protein-derived antagonist

Human parathyroid hormone-related protein (PTHrP) is expressed in various tissues where it acts as an endocrine/paracrine factor involved in cellular growth, differentiation and development of fetal skeleton. As for parathyroid hormone (PTH), which is the hormone responsible for regulation of extracellular calcium homeostasis, the N-terminal 1-34 fragment can reproduce the full spectrum of calciotropic activities inherent in full-length PTH. Truncation of six amino acid residues from the N-terminus of both hormone sequences generates 7-34 fragments which act as weak antagonists. Although PTH(7-34) is a pure antagonist, PTHrP(7-34) acts as partial agonist against the receptor shared by both hormones, the PTH/PTHrP receptor. In the current study, we analyzed the conformation of [Leu¹¹,d -Trp¹²,Lys²⁶,Asp³⁰]PTHrP(7-34)NH₂ (hybrid-lactam) in a 1:1 mixture of H₂O/TFE-d₃ at pH 4 by circular dichroism, nuclear magnetic resonance and distance geometry calculations. This weak antagonist (K_b= 650 nm ) combines two modifications: Leu¹¹,d -Trp¹² (K_b= 5.1 nm ), reported to eliminate partial agonism and enhance potency, and Lys²⁶-Asp³⁰ lactamization (K_b= 31 nm ), aimed to stabilize the helical structure of the principal binding domain attributed to residues 25-34. The helical content in 30% trifluoroethanol is 88%, i.e., higher than the corresponding linear analog, and comprises the d -Trp¹²-Thr³³ segment. This hybrid lactam contains a rigid helical segment spanning the 14-18 sequence followed by a hinge motif around Arg^19-20, but the sequence 14-18 forms a stable helix. In all potent lactam-containing, PTHrP-derived agonists and antagonists studied so far, the dominant structural motif consists of two helical domains at the two ends of the sequence and of two hinge regions centered around Gly¹²-Lys¹³ and Arg¹⁹. The weakly active agonists and antagonists do not exhibit the “hinge” around position 19. These findings suggest that the presence and location of discrete hinge regions that connect the N- and C-terminal helices are essential for generating the bioactive conformation of ligands for the PTH/ PTHrP receptor.     

Effects of adrenomedullin,C-type natriuretic peptide,and parathyroid hormone-related peptide on calcification in cultured rat vascular smooth muscle cells

To clarify the regulating mechanism of vascular calcification, the investigators observed the effects of three vasoactive peptides, adrenomedullin (ADM), C-type natriuretic peptide (CNP), and parathyroid hormone-related peptide (PTHrP) on calcification in rat vascular smooth muscle cells (VSMCs). Beta-glycerophosphate stimulated growth and calcification in VSMCs. Adrenomedullin and CNP lowered beta-glycerophosphate-induced increase in VSMC growth. All three vasoactive peptides attenuated the increases of 45Ca accumulation, calcium content, and alkaline phosphatase activity in calcified VSMCs. As for comparing the inhibitory effects, the strongest was PTHrP. Both ADM and PTHrP increased cyclic adenosine monophosphate (cAMP) content in calcified VSMCs, but CNP upregulated cyclic guanosine monophosphate (cGMP) content. The PKA inhibitor PKAI completely reversed the inhibition of ADM on cell growth and all inhibitory effects of PTHrP on the parameters of calcification. The PKG inhibitor H8, however, strongly antagonized all the inhibitory effects of CNP on calcification. These data suggested that beta-glycerophosphate-induced calcification in VSMCs was inhibited by ADM, CNP, and PTHrP. Adrenomedullin and PTHrP inhibited VSMC calcification partially through the cAMP/PKA pathway, whereas CNP inhibited VSMC calcification through the cGMP/PKG pathway. This study could be of help in understanding the pathogenesis of vascular calcification, and providing new target for clinical treatment of cardiovascular diseases associated with vascular calcification.     

PTEN蛋白在膀胱癌组织中的表达

目的 探讨PTEN蛋白在膀胱癌组织中的表达及意义.方法 以51例膀胱癌石蜡包埋组织为研究对象,采用免疫组织化学S-P法观察PTEN蛋白在膀胱癌组织中的表达.结果 51例膀胱癌组织中PTEN蛋白阳性率为80.39%（41/51）,PTEN蛋白在膀胱癌组织与正常膀胱组织之间阳性率表达差异无显著性（P＞0.05）;膀胱癌组织中PTEN蛋白与肿瘤的分期、分级、淋巴结转移以及复发无关（P＞0.05）.结论 PTEN蛋白在膀胱癌组织与正常膀胱组织之间的阳性率表达差异无显著性;膀胱癌组织中PTEN蛋白与肿瘤的分期、分级、淋巴结转移以及复发无关.提示PTEN蛋白可能未参与膀胱癌的发生     

食管癌组织中复制蛋白A的表达

目的 探讨食骨鳞癌组织中复制蛋白A(RPA)的表达及临床意义.方法 收集食管癌根治手术切除标本40例,病理均为鳞状细胞癌.用免疫组织化学法检测食管癌组织及癌旁正常组织中RPA1和RPA2的表达.结果 食管癌组织RPA1表达阳性率为65.25%,明显高于癌旁正常组织的35.65%(P<0.01);癌组织RPA2表达阳性率58.90%,亦明显高于癌旁组织的16.71%(P<0.01).结论 食管癌组织中RPA1、RPA2的表达明显高于同一标本的癌旁正常组织.RPA1、RPA2可能在食管癌的发生、发展过程中起一定的作用.     

KLK14在宫颈癌组织中的表达及其意义

目的探讨KLK14蛋白宫颈癌中的表达及与临床病理关系。方法应用免疫组织化学法SP检测10例正常宫颈14例慢性宫颈炎48宫颈癌组织中KLK14蛋白的表达。应用半定量逆转录聚合酶链反应法RT-PCR检测25例宫颈鳞癌及10例正常宫颈组织中KLK14 mRNA的表达。结果KLK14蛋白在48例宫颈癌中阳性率72．9％,正常宫颈20％,慢性宫颈炎14．3％,三者相比差异有统计学意义,KLK14蛋白阳性率与肿瘤临床分期,病理学相关（P〈0．05）,与年龄组织学分级淋巴转移无关（P〉0．05）。KLK14 mRNA在宫颈癌相对表达量高于正常宫颈组织。结论KLK14表达参与宫颈癌的发生发展。     

P16 蛋白在鼻咽癌中的表达研究

目的研究P16蛋白在鼻咽癌中的表达情况及其与鼻咽癌临床病理特征的关系。方法采用免疫组织化学链霉菌抗生物素蛋白．过氧化物酶连接法检测48例鼻咽癌和16例正常鼻咽黏膜上皮组织中P16蛋白的表达情况,分析鼻咽癌患者中P16蛋白表达与鼻咽癌临床病理特征之间的关系。结果鼻咽癌中P16蛋白表达的缺失率为70．8％（34／48）;鼻咽正常黏膜上皮组织为37．5％（6／16）,二者缺失率比较,差异有统计学意义（Х^2=18．364,P=0.000）;P16蛋白表达缺失率在低分化鼻咽癌中为75．0％（30／40）,在中分化鼻咽癌中为50．0％（4／8）,二者比较,差异有统计学意义（Х^2=11．308,P=0.010）。P16蛋白表达与鼻咽癌临床分期、淋巴结转移、远处转移及复发呈负相关（P〈0．05）。结论P16蛋白对于提高鼻咽癌早期诊断的有效性和准确性有着重要作用,值得进一步研究。     

乳糖诱导表达重组人甲状旁腺激素rhPTH(1-34)

目的研究以乳糖代替异丙基硫代半乳糖苷(IPTG)诱导表达重组人甲状旁腺激素的可行性。方法对乳糖浓度、诱导时间及发酵培养方式进行研究,确定了乳糖诱导的最佳条件。结果 0.6%的乳糖诱导4 h,目的蛋白的表达量约占菌体总蛋白的56.8%,采取分批补料培养方式,5 L发酵罐中蛋白表达量达到42.6%。结论乳糖能作为诱导剂诱导rhPTH(1-34)在大肠杆菌中的表达。     

Pokemon蛋白和p53蛋白在卵巢癌组织中的表达及其意义

目的探讨卵巢癌组织中Pokemon蛋白和p53蛋白表达,明确其与临床病理的关系。方法选取52例卵巢癌组织作为观察组,60例卵巢良性病变组织作为对照组。免疫组化测定两组组织中Pokemon蛋白和p53蛋白的表达。结果观察组和对照组中Pokemon蛋白阳性率分别为69．2％（36／52）和11．7％（7／60）,差异有统计学意义（χ2=12．74,P〈0．01）。观察组和对照组中p53蛋白阳性率分别为78．8％（41／52）和15．0％（9／60）,差异有统计学意义（χ2=22．71,P〈0．01）。Pokemon蛋白表达与卵巢癌患者的肿瘤浸润深度（χ2=9．16,P〈0．05）、临床分期（χ2=5．86,P〈0．05）、淋巴结是否转移（χ2=32．28,P〈0．01）和是否复发（χ2=27．08,P〈0．01）均有显著的关系,与肿瘤分化程度（χ2=0．82,P〉0．05）无明显关系。p53蛋白表达与卵巢癌患者的肿瘤浸润深度（χ2=8．24,P〈0．05）、临床分期（χ2=9．24,P〈0．05）、淋巴结是否转移（χ2=19．62,P〈0．01）和是否复发（χ2=21．16,P〈0．01）均有显著的关系,与肿瘤分化程度（χ2=0．77,P〉0．05）无明显关系。经相关性分析,Pokemon蛋白和p53蛋白阳性表达有明显正相关关系（r=0．297,P〈0．05）。结论Pokemon蛋白和p53蛋白与卵巢癌的诊断、病变程度和预后均有密切关系。     

Twist蛋白上皮性钙黏蛋白mRNA及蛋白在喉鳞癌中表达的研究

目的 观察Twist蛋白、上皮性钙黏蛋白mRNA及蛋白在喉鳞癌组织中的表达及其与临床病理特征的关系,并探讨其在喉鳞癌发生、发展中的作用及临床应用价值.方法 应用原位分子杂交检测上皮性钙黏蛋白mRNA和免疫组织化学SP法检测Twist蛋白、上皮性钙黏蛋白分别在80例喉鳞癌、30例不典型增生和20例慢性炎症组织中的表达.结...     

食管鳞癌组织中FAK蛋白的表达

目的研究食管鳞癌组织中黏着斑激酶(FAK)蛋白的表达及其与临床病理参数的关系。方法采用Western blot法检测46例食管鳞癌组织及其癌旁正常组织中FAK蛋白的表达,分析FAK蛋白的表达与临床参数的相关性。结果癌组织中FAK蛋白的表达水平高于癌旁正常组织(P<0.01)。低分化癌组织中FAK的表达量较高、中分化组织中高,有淋巴结转移的较无转移的表达高(P<0.05)。结论 FAK蛋白在食管鳞癌组织中高表达,其表达量与食管鳞癌的分化程度、浸润深度和淋巴结转移有关。FAK表达的高低有可能作为食管癌预后判断的一种新指标。