Endostatin, a collagen XVIII fragment, is a potent anti-angiogenic protein. We sought to identify its endothelial cell surface receptor(s). Alkaline phosphatase- tagged endostatin bound endothelial cells revealing two binding affinities. Expression cloning identified glypican, a cell surface proteoglycan as the lower-affinity receptor. Biochemical and genetic studies indicated that glypicans' heparan sulfate glycosaminoglycans were critical for endostatin binding. Furthermore, endostatin selected a specific octasulfated hexasaccharide from a sequence in heparin. We have also demonstrated a role for endostatin in renal tubular cell branching morphogenesis and show that glypicans serve as low-affinity receptors for endostatin in these cells, as in endothelial cells. Finally, antisense experiments suggest the critical importance of glypicans in mediating endostatin activities.

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Transforming growth factor-␤ (TGF-␤) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-␤ signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitinproteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-␤ and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitinmediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-␤-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-␤ signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation.

In the present study, we examined the expression and the localization of apamin-sensitive small conductance Ca(2+)-activated K(+) channels (SK channels) in the mouse intestine. SK3-immunoreactivity (IR) was detected in both ileum and colon. Double staining experiments showed that SK3-IR was colocalized with prolyl 4-hydroxylase (PH(alpha))-IR, but not with c-Kit-IR which are markers of fibroblast cells and the interstitial cells of Cajal (ICC), respectively. Although SK3-IR was colocalized with vimentin-IR, which is another marker of ICC, the reactivity of SK3-immunopositive cells was weaker than that of ICC. The SK3-immunopositive cells were similarly present in the intestine of c-Kit mutant mice (W/W(V)), in which ICC were absent, and its wild-type mice. The immuno-electron microscopic analysis indicated that SK3 was localized in the cells that had some similar morphological features to ICC, but obviously different from ICC. The SK3-immunopositive cells had gap junctions with the smooth muscle cells. The gap junctions were smaller than those between ICC and smooth muscle cells. These results indicate expression of SK3 in fibroblast-like cells, but not in ICC, and suggest participation of the cells in the intestinal motility.

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