The goal of this proposal is to determine the role in the pathogenesis of the disease mastocytosis played by the c-KIT proto-oncogene, the KIT receptor tyrosine kinase that it encodes, and the ligand for KIT. Cutaneous mastocytosis involves mast cells melanocytes, two cell types which express KIT and respond to the KIT ligand(KL). Because activation of KIT can cause neoplastic transformation, abnormalities of KIT and KL were sought in patients with mastocytosis. Targeted sequencing a short portion of c-KIT cDNAs derived from mast cells showed a mutation know to result in constitutive activation of KIT in three of six patients with different forms of mastocytosis. KL, which is mostly membrane bound in normal skin, was found in a soluble form in lesions of cutaneous mastocytosis. It was also discovered that the mast cell enzyme chymase could specifically cleave KL to produce bioactive soluble KL (sKL). The proposed research will test the hypothesis that mast cell proliferation in mastocytosis is initiated by activating c-KIT mutations, and that mast cell chymase or other enzyme cleave membrane bound KL (mKL) from stromal cells in cutaneous mastocytosis, releasing sKL which may activate melanocytes and stimulate further mast cell proliferations. The strategy is to determine the frequency of activating c-KIT mutations in different forms of mastocytosis, the ways in which sKL is generated in the skin, and the ability of c-KIT mutations to cause mastocytosis in transgenic mice.
The specific aims are:
Aim 1 : To determine the frequency of activating c-KIT mutations in different forms of mastocytosis. cDNAs that include the entire coding region of c-KIT will be derived from lesions of cutaneous mastocytosis and sequenced. cDNAs with novels mutations will be subcloned into mammalian expression vectors and expressed in vitro to determine their effects on KIT activation.
Aim 2 : To identify inflammatory cell enzymes than cleave KL and produce biologically active sKL. Preparations of purified recombinant KL or mKL will be co-incubated with tryptase, elastase, and cathepsin. Cleavage products will be identified by western blotting and protein sequencing, and their bioactivity determined by melanocyte-KIT phosphorylation assays.
Aim 3 : To reproduce mastocytosis intransgenic mice. To test the hypothesis that c-KIT mutations can cause mastocytosis, the chymase promoter will be used to express mutated KIT in mast cells of transgenic mice the occurrence of mastocytosis in these animals will show that c-KIT mutation can cause mastocytosis.
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