PARATHYROID HORMONE-LIKE PEPTIDE FROM KERATINOCYTES

体外正常人角质形成细胞产生一种分子， 甲状旁腺激素受体，并刺激积累 大鼠骨肉瘤细胞中的环磷酸腺苷（cAMP）。 述分子不是 甲状旁腺激素本身，但与一组 甲状旁腺激素样多肽与体液免疫临床综合征的关系 恶性高钙血症。 这项提案的目的是净化 角质形成细胞因子，确定其结构，并表征其 生物活动。 该项目有三个部分： 因素：第一个目标是确定最大限度地提高 人角质形成细胞产生的PTH样因子。 最终目标是 以在角质形成细胞条件下产生至少10 μ g/周的因子 介质 纯化和生化表征：纯化方案 与已经描述的肿瘤衍生的PTH样因子类似， 用于净化。 简而言之，这采用了一系列步骤 包括通过超滤、分子筛 色谱法，疏水作用色谱法，等电 聚焦和高效液相色谱法。 高度纯化 材料将被用作免疫原，并且当足够量的 均匀的材料是可用的，我们将获得部分氨基酸 顺序 生物活性：虽然对生物活性的确定性研究， 这种角质形成细胞因子必须等待其纯化， PTH样活性使我们能够产生可检验的假设。 因此我们 假设角质形成细胞因子应该具有显著的局部或 由于其与PTH结合的能力， 受体，增加细胞内cAMP。 我们计划测试一下 通过检查PTH对角质形成细胞生长的影响， 分化以及纯化的KCM因子对其他 PTH敏感系统，如体外骨吸收。 本提案的长期目标是：1）更好地了解 角质形成细胞稳态和局部产生的生物学作用 角质形成细胞稳态的常见病症中的活性分子，例如 银屑病; 2）更好地了解外界因素如何产生 甲状旁腺调节钙和维生素D代谢， 疾病

Normal human keratinocytes in vitro make a molecule that appears to bind to the parathyroid hormone receptor and stimulates the accumulation of cyclic-AMP (cAMP) in rat osteosarcoma cells. The molecule is not parathyroid hormone itself, but shares many properties with one group of PTH-like polypeptides responsible for the clinical syndrome of humoral hypercalcemia of malignancy (HHM). The aims of this proposal are to purify the keratinocyte factor, determine its structure, and characterize its biological activities. The project has three parts: Production of the factor: The first goal will be to define culture conditions which maximize human keratinocyte production of the PTH-like factor. The ultimate goal is to produce at least 10 Mug/week of the factor in keratinocyte-conditioned medium. Purification and biochemical characterization: A purification scheme similar to that already described for tumor-derived PTH-like factors will be used for purification. In brief, this employs a series of steps including concentration by ultra-filtration, molecular sieve chromatography, hydrophobic interaction chromatography, isoelectric focusing, and high-performance liquid chromatography. Highly purified material will be used as immunogen, and when sufficient amounts of homogeneous material are available, we will obtain a partial amino acid sequence. Biological activity: While definitive studies of the biological action of this keratinocyte factor must await its purification, knowledge of its PTH-like activity allows us to generate testable hypotheses. Thus, we hypothesize that the keratinocyte factor should have significant local or distant biological effects by virtue of its ability to bind to the PTH receptor and increase intracellular cyclic-AMP. We plan to test this hypothesis by examining the effect of PTH on keratinocyte growth and differentiation as well as the effect of purified KCM factor on other PTH-sensitive systems such as in vitro bone resorption. Long-range goals of this proposal are: 1) to better understand keratinocyte homeostasis and the role of locally produced, biologically active molecules in common disorders of keratinocyte homeostasis, such as psoriasis; 2) to better understand how factors produced outside the parathyroid gland modulate calcium and Vitamin D metabolism in health and disease.