Fibronectin-Induced Proliferation in Thyroid Cells Is Mediated by αvβ3 Integrin through Ras/Raf-1/MEK/ERK and Calcium/CaMKII Signals

Abstract

We recently demonstrated in an immortalized thyroid cell line that integrin stimulation by fibronectin (FN) simultaneously activates two signaling pathways: Ras/Raf/MAPK kinase (Mek)/Erk and calcium Ca2+/calcium calmodulin-dependent kinase II (CaMKII). Both signals are necessary to stimulate Erk phosphorylation because CaMKII modulates Ras-induced Raf-1 activity. In this study we present evidence that extends these findings to normal human thyroid cells in primary culture, demonstrating its biological significance in a more physiological cell model. In normal thyroid cells, immobilized FN-induced activation of p21Ras and Erk phosphorylation. This pathway was responsible for FN-induced cell proliferation. Concurrent increase of intracellular Ca2+ concentration and CaMKII activation was observed. Both induction of p21Ras activity and increase of intracellular Ca2+ concentration were mediated by FN binding to alphavbeta3 integrin. Inhibition of the Ca2+/CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2+/CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered.

Activation of tumor cell integrin α v β 3 controls angiogenesis and metastatic growth in the brain

“…However, in our model, tumor cell survival was not limiting, and we found increased proliferation but no evidence for enhanced survival of tumor cells upon ␣ v ␤ 3 activation in normoxic brain lesions. Integrins can signal through different growth promoting pathways, including Src and MAPK (27)(28)(29)(30). However, we detected no significant differences in pSrc and pERK levels between ␤ 3 WT-and ␤ 3 D723R-expressing brain lesions (not shown).…”

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

“…In other cell types, endogenous Raf-1, but not B-Raf, formed a complex with activated CaMKII that was precipitated by antiCaMKII antibodies and recognized by anti-Raf-1 antibodies. 12,13 The selective CaMKII/Raf isoforms interaction was confirmed in COS-7 (Fig. 2).…”

“…Co-immunoprecipitation experiments showed that activated CaMKII interacts with Raf-1 in vivo, and that the complex CaMKII/Raf-1 is necessary for ERK activation from different stimuli. [12][13][14]16 This role of CaMKII in the ERK pathway appears to be a widespread mechanism, as it occurs upon different stimuli (fibronectin-stimulated integrins, serum, insulin, RET/PTC oncogene, Ras V12 ), in a number of different cell types (thyroid cells, fibroblasts, L6 myotubes, Hep3B, NIH-3T3 and COS-7 cells) with few exceptions (colon adenocarcinoma cells LoVo and prostate cancer cells DU-145, data not shown). Nevertheless, the physiological role of the CaMKII/ Raf-1 interplay is likely cell context-dependent, as it participates in and is modulated by the complex crosstalk of signal transduction pathways existing in any living cell.…”

“…28 Our previous results did not support a significant role for B-Raf in the signal generated by FN-integrin binding in thyroid cells. 13 Experiments shown in Figure 3B and C were replicated, and B-Raf activation was determined by kinase assay. Neither a reduction of Ras V12 -stimulated B-Raf activation by CaMKII inhibition, nor B-Raf activity stimulation by active CaMKII was observed (data not shown).…”

“…[12][13][14][15][16] CaMKII is an ubiquitous serine/ threonine kinase whose activation is modulated by intracellular whether Ras stimulates CaMKII phosphorylation at Thr286, NIH-3T3 cells were transiently transfected with expression vectors encoding HRas V12 and KRas V12 isoforms. 48 h post-transfection, serum-starved cells were treated for 30 min with the calmodulin inhibitors W7 or TFP, and CaMKII phosphorylation at Thr286 was visualized by western blot using a phosphospecific antibody (Fig.…”