Among candidate compounds in this path are the tyrosine phosphatase Shp2 and the adaptor molecule Gab TGF-beta 1. In Fig. 6A,B, we examined the power of HGF and IL 6 to induce phosphorylation of Gab1 and Shp2 in ANBL 6 cells. Because these cells develop HGF endoge nously resulting in minimal c Met expression, we preincubated the cells over night with anti HGF serum to improve c Met expression before addition of IL 6 for 10 min with or without the existence of the c Met kinase inhibitor as indicated in Fig. 6A,B.
IL 6 caused minimal phosphorylation of tyrosine 542 on Shp2 under these conditions. In contrast, HGF induced low but detectable phosphorylation of Gab1. Importantly, in the clear presence of HGF, the phosphorylation of Shp2 was further increased with IL 6. More over, the Gab1 and Shp2 phosphorylation induced with the mixture of HGF and IL 6 was substantially reduced in the current presence of the d Met kinase inhibitor. These results suggest that the mix of HGF and IL 6 gave more pronounced activation of Shp2 than class II HDAC inhibitor both cytokine alone, suggesting that Shp2 activation induced by IL 6 is also dependent on h Met activation. IL 6 has been reported to phosphorylate the IGF 1 receptor as foundation for synergy between IL 6 and IGF 1.
Phosphorylation of c Met activated by IL 6 could have been an explanation for potentiation of Shp2 phosphorylation in ANBL 6 cells. But, this appeared to not function as case. We tried the result of the story Shp2 inhibitor NSC 87877, to see if Shp2 activation was involved in activation of p44 42 MAPK activation. This inhibitor binds to the catalytic cleft of Shp2 and inhibits both basal, and EGF induced Shp2 phosphatase activity as well as EGFinduced p44 42 MAPK Organism phosphorylation which is considered to be influenced by Shp2. In the presence of IL 6 and endogenous HGF, NSC 87877 inhibited phosphorylation of p44 42 MAPK in ANBL 6 cells in a dose dependent fashion, without affecting the phosphorylation of STAT3.
These results claim that whereas Shp2 is involved with p44 42 MAPK activation, it has no purpose in phosphorylation which can be totally determined by IL 6 in this environment. Moreover, the synergy noticed in Ras MAPK signaling would depend on the synergy in phosphatase activity of Shp2. The key nding reported here is that IL 6 caused growth may be influenced by c Met signaling in myeloma cells.
The potentiating effectation of HGF c Met on IL 6 signaling could be explained by two mechanisms: IL 6 increased the degree of c Met on the cell surface of myeloma cells making cells more sensitive and painful to HGF, and IL 6 counted on HGF c Met to totally BI-1356 activate the RasMAPK process possibly through Shp2 activation. HGF is situated in bone marrow plasma of both healthy subjects and myeloma clients, and bone marrow stromal cells constitutively produce HGF. Moreover, syndecan 1 binds HGF on top of myeloma cells bringing HGF in close proximity of its receptor c Met.