TGFBIp mediated apoptosis: A role in cancer and diabetes mellitus type II
TGFBIp is an extracellular matrix protein that is upregulated by TGF-beta1 and expressed during embryonic development, and in adult tissues. TGFBIp's primary sequence designates it as a member of the fasciclin family of cell-surface and secreted proteins, principally because TGFBIp has four internal fasciclin-like repeats. Seven integrin-binding and two glycosaminoglycan-binding sequences are within the fasciclin-like domains and the C-terminus. Protein characterization revealed that TGFBIp's C-terminus is fragmented, yielding a mature TGFBIp lacking 10 kDa protein, including regions that encode the integrin-binding sites RGD and EPDIM, both of which have been implicated in TGFBIp-induced apoptosis. Recently, we have shown that TGFBIp induced apoptosis in osteosarcoma cells. A significant increase in apoptosis can be brought about by treating cells with TGF-beta1 to induce TGFBIp expression, by cells overexpressing recombinant TGFBIp, and by adding-back TGFBIp to cell growth medium. The apoptotic signal was localized to TGFBIp's extreme C-terminal 69 amino acid residues. This study extends the findings, showing that the integrin-binding sequence EPDIM in the C-terminus is the apoptotic signal for osteosarcoma cells. Mutating the signal to EPAIM significantly blocked apoptosis (p<0.001). In addition, the studies show that the low levels of TGFBIp expression in the absence of exogenous TGF-beta1 is sufficient to account for baseline apoptosis in cultured osteosarcoma cells. Stable shRNA-knockdown of osteosarcoma TGFBIp resulted in only 13% apoptosis as compared to 32% in control cells. These findings indicate that TGFBIp plays a fundamental role in apoptosis. Indeed, TGFBIp's apoptotic effect raised the question of whether TGFBIp-induced apoptosis plays roles in diseases hallmarked by TGF-beta1. In this venue, to complement previous studies, the role of TGFBIp in diabetes mellitus type II (DMT2) was investigated. TGFBIp is overexpressed in DMT2 kidney, and apoptosis is implicated in renal failure, therefore common denominators of tumor progression and DMT2 are TGFBIp and apoptosis. With this perspective, and considering the results of the tumor studies, human kidney cortex from DMT2 and non-DMT2 donors was tested for TGFBIp expression. TGFBIp immunohistochemical and TUNEL analyses clearly showed co-localization of TGFBIp and TUNEL-positive cells in proximal tubules in DMT2 cortex, suggesting that tubular segments are the areas undergoing TGFBIp-mediated damage in DMT2. TGFBIp quantity in DMT2 was 10-fold more than that in control cortex, and TGFBIp isolated from DMT2 cortex resolved on polyacrylamide gels as three bands consistent with C-terminal fragmented TGFBIp, indicating that particularly renal proximal tubule epithelial (RPTE) cells are subject to TGFBIp-mediated apoptosis. To test this possibility, human RPTE early-passage cells were cultured in growth medium containing different concentrations of TGFBIp. Cell apoptosis was found to be dependent on TGFBIp concentration in the medium. TGFBIp concentrations of 5 mug/ml induced a significant increase in the number of apoptotic cells (p<0.001) and activated caspase-3. Caspase-3 specific activity was 5.3 pM/min/cell, a 23-fold increase over caspase-3 in nontreated cells. These data demonstrate that TGFBIp-induced apoptosis is caspase-3 mediated. Increase in blood glucose is a manifestation of DMT2, and it is reported that TGFBIp is induced by high glucose. Increased glucose concentrations in RPTE cell growth medium resulted in significant apoptosis that was blocked by anti-TGFBIp antibody (p<0.001), strongly implicating TGFBIp in the pathway. Taken together, these studies point out the role of TGFBIp as an important and key molecule mediating effects of TGF-beta1. In the case of tumor progression, TGFBIp plays a beneficial role prompting suppression of tumor growth, presumably through apoptosis. In the sense of DMT2, TGFBIp contributes to RPTE cell apoptosis, thereby leading to renal failure. Understanding these mechanisms, particularly TGFBIp expression, fragmentation and the TGFBIp-induced apoptotic pathway is expected to lead to development of novel treatments for various diseases including cancer and diabetes.