|Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
(1) Fibroblast Growth Factor(FGF) is one of the key molecules for the growth and differentiation of gastrointestinal epithelial cells. FGF acts through binding to FGF receptor (FGFR), its specific high-affinity transmembrance receptors. Four closely related members of FGF receptor family have been currently known and have common features such as three extracellular immunoglobulin-life domains, single transmembrane portion and intracellular split tyrosine-kinase domain. FGFR 1,2 ＆3 have alternative splicing form encoded by separate exons for the C-terminal half of the third Iglike domain (called IIIb and IIIc type), but FGF receptor 4 tow no such separate exons. Furthermore, FGFR 1,2 ＆3 have another splice variant form of non-transmernbrane type, however such a variant form has not been reported for FGFR 4.
We have detected a non-transmembrane type receptor of human FGFR 4 in the intestinal epithelial cell lines such as 'Intestine 407' and 'Caco-2' by RT-PCR. Sequence analysis of this re
ceptor revealed that (1) exon 9 coding the single transmembrane portion is. deleted and replaced by 'Ihtron 9', The variant form is 120 bps shorter than wild type and has 24 amino acids including hydrophilic ones. (2) Signal sequences in Exon 2 are maintained These two findings suggested that this variant receptor might be a soluble form such as K-sam. The microcapillary-used single cell RTPCR showed this soluble receptor is co-expressed with the transmernbrane-type receptor in the single cell. Western blot analysis using human FGFR 4 antibody revealed that this receptor was secreted from the intestinal cell lines in the serum-free medium. Furthermore, this soluble receptor is expressed in gastric cancer cell line KATO-III ; pancreatic cancer cell line ASPC- 1, and human normal mucosal specimens of esophagaus, stomach and colon.
(2) Paneth cells are morphologically well-characterized granule-containing cells which are localized in the bases of intestinal crypts. Their cytoplasmic granules contain large amounts of zinc. our previous study showed that a single intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, to rats induced precipitation of zinc-dithizonate complexes in the cytoplasm and selective killing of Panethe cells followed by a prominent transient wave of cell proliferation in entire intestinal crypts.
Various growth factors and cytokines such as EGF, TGF-alpha, HGF and FGF have been shown to be involved in the modulations of epithelial cell regeneration, intestine using this experimental model. One of the DNA sequence from Caco-2 showed full-length rat thioredoxin cDNA . To confirm the growth promoting effect of rat thioredoxin, we transfected full-length thioredoxin cDNA into Caco-2 cells again, and obtained several stable clones overexpessing thioredoxin. These clones revealed the proliferation even in the medium containing only 1 % FBS or containing oxidative reagent, Diamide 100μM or Hydrogen Peroxide 400μM, although the control clones did not show any growth.
These results show that thioredoxin could be a key molecule to regulate the small intestinal epithelial regeneration and proliferation. Less