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.
