Glucose Regulation of Integrin-Associated Protein Cleavage Controls the Response of Vascular Smooth Muscle Cells to Insulin-Like Growth Factor-I

Abstract

Vascular smooth muscle cells (SMC) maintained in high glucose are more responsive to IGF-I than SMC maintained in normal glucose due to a difference in the Shc phosphorylation response. In this study we aimed to determine the mechanism by which glucose regulates the sensitivity of SMC to IGF-I. For Shc to be phosphorylated in response to IGF-I it must be recruited to tyrosine-phosphorylated sites on Src homology 2 domain-containing phosphatase (SHP) substrate-1 (SHPS-1). The association of integrin-associated protein (IAP) with SHPS-1 is required for SHPS-1 tyrosine phosphorylation. When SMC were grown in 5 mm glucose, the amount of intact IAP was reduced, compared with SMC grown in 25 mm glucose. This reduction was due to proteolytic cleavage of IAP. Proteolysis of IAP resulted in loss of its SHPS-1 binding site, which led to loss of SHPS-1 phosphorylation. Analysis of the conditioned medium showed that there was more protease activity in the medium from SMC cultured in 5 mm glucose as compared with 25 mm. Inhibition of matrix metalloprotease-2 synthesis using RNA interference or its activity using a specific protease inhibitor protected IAP from cleavage. This protection was associated with an increase in IAP-SHPS-1 association, increased recruitment and phosphorylation of Shc, and increased cell growth in response to IGF-I. Our results show that the enhanced response of SMC in 25 mm glucose to IGF-I is due to the protection of IAP from proteolytic degradation, thereby increasing its association with SHPS-1 and allowing the formation of the SHPS-1-Shc signaling complex.

Signal regulatory protein alpha (SIRPα)/CD47 interaction and function

“…The use of heterozygous CD47−/+ mice showed that the level of CD47 on red blood cells was also critical in determining their phagocytosis if they were opsonised but not with unopsonised cells [36]. Another interesting result from neural cells on levels of expression is the finding that CD47 can be released from the cell surface by proteolysis [37]. One point that is not resolved in this study is where the cleavage occurs?…”

Integrin-Associated Protein Association With Src Homology 2 Domain Containing Tyrosine Phosphatase Substrate 1 Regulates IGF-I Signaling In Vivo

“…In order for SHPS-1 to be phosphorylated, it must be associated with IAP (12). Our recent in vitro studies have determined that in SMCs cultured in 5 mmol/l glucose, SHPS-1 phosphorylation and formation of the SHPS-1-SHP-2-c-Src-Shc signaling complex (which is required for cellular replication) is impaired because of constitutive cleavage of IAP, which disrupts SHPS-1 binding (14). When the status of IAP in the aortic homogenates was determined, there was a significant (fivefold) increase in intact IAP detected in the homogenates from the hyperglycemic mice compared with controls.…”

“…The anti-IAP monoclonal antibody, B6H12, was purified from a cell line derived from a B-cell hybridoma (13). The anti-IAP antibody (referred to as R569), which recognizes amino acids 41 and 61 in the extracellular domain of IAP, has been described previously (14). Induction of hyperglycemia in mice.…”

“…Analysis of MMP-2 gelatinase activity by zymography. MMP-2 gelatinase activity was assessed in aorta homogenates and serum-free conditioned medium from murine aortic SMCs (mSMCs) by gelatin zymography (14). Isolation of mSMCs.…”

“…mSMCs were isolated from the aorta of wild-type C57/B6 male mice and maintained in medium containing either 5 or 25 mmol/l glucose (16). Medium containing 5 mmol/l glucose was supplemented to 25 mmol/l with mannitol (the addition of mannitol does not affect IAP cleavage when SMCs are grown in 5 mmol/l glucose [14]). Both types of medium contained 1.0 mmol/l pyruvate.…”