The ability of protein tyrosine phosphatase SHP-1 to suppress NFkappaB can be inhibited by dominant negative mutant of SIRPalpha

In contrast with hematopoietic cells and fibroblasts, which express mainly one form of protein tyrosine phosphatase (PTP) SHP-1 or SHP-2, epithelial cells like A431, HeLa, and 293 express both forms of PTP. These two PTP regulate NFkappaB activity differently; SHP-1 inhibits and SHP-2 stimulates NFkappaB activation. In epithelial cells the process of NFkappaB activation depends on the combination of two PTP activities. The activity of PTP SHP-1 dominates in this tandem according to our data. The signal regulatory protein (SIRPalpha) is the adapter and the substrate of PTP SHP-1 and SHP-2. We investigated the role of SIRPalpha and its dominant negative mutant in PTP activities in 293 cells. The overexpression of wild-type SIRPalpha suppresses the activities of both PTP, but has a stronger effect on PTP SHP-2, especially when this protein is overexpressed in 293 cells. In contrast with wild-type SIRPalpha, its dominant negative mutant acts predominantly against PTP SHP-1, and can be detected in the complex with PTP SHP-1. The expression of dominant negative mutant of SIRPalpha has an effect similar to the expression of dominant negative PTP SHP-1 in the process of NFkappaB activation.     

Differential activation of the SMalpha A promoter in smooth vs. skeletal muscle cells by bHLH factors

E-box/basichelix-loop-helix (bHLH)-dependent regulation of promoters for skeletalmuscle-specific genes is well established, but similar regulation ofsmooth muscle-selective promoters has not been reported. Usingtransient transfection assays of smooth muscle -actin (SMA)promoter-chloramphenicol acetyltransferase (CAT) reporter constructs inrat vascular smooth muscle cells (SMCs) and L6 skeletal myotubes, weidentified two activator elements, smE1 and smE2, with sequencescorresponding to E-box (5'-CAnnTG-3') motifs. In L6myotubes, 4-bp mutations of smE1 or smE2 E-box motif alone completelyabolished promoter activity. In contrast, mutation of smE1 and smE2 wasrequired to reduce promoter activity in SMCs. Supershift analysesidentified a myogenin-containing complex as the predominant smE1 andsmE2 binding activity in skeletal muscle, and myogenin overexpressiontransactivated the promoter. Supershift analyses with SMC extractsdemonstrated that the bHLH protein upstream stimulatory factor (USF)bound smE1, and USF overexpression transactivated the promoter in ansmE1-dependent manner. In summary, our results provide novel evidenceimplicating E-box elements in directing expression of the SMApromoter through distinct bHLH factor complexes in skeletal vs. smooth muscle.