Soung Hoo Jeon

Both ERK and Wnt/β-catenin pathways are involved in Wnt3a-induced proliferation

The Wnt family of proteins regulates development and cell growth. We identified Wnt3a-based regulatory mechanisms for cell proliferation in NIH3T3 fibroblast cells. The degree of Wnt3a-induced proliferation was reduced by β-catenin small interfering RNA (siRNA) and extracellular signal-regulated kinase (ERK) siRNA, indicating that both the ERK and Wnt/β-catenin pathways are involved in Wnt3a-induced proliferation. Wnt3a immediately and transiently activated the Raf-1-MEK-ERK cascade in a manner distinct from that of the β-catenin increase seen in cells treated with Wnt3a. Wnt3a-induced ERK activation was maintained even though basal ERK activities were reduced by β-catenin siRNA, indicating that Wnt3a may activate the ERK pathway independently of β-catenin. The ERK pathway was however, activated by β-catenin transfection, which was abolished by co-transfection with dominant-negative Tcf-4. Therefore, ERK pathway activation by Wnt signaling could occur at multiple levels, including β-catenin-independent direct signaling resulting from a Wnt3a and β-catenin/Tcf-4-dependent post gene transcriptional event. Wnt3a stimulated the G1 to S phase cell cycle progression. This stimulation was reduced by the ERK pathway inhibitor, indicating that Wnt3a promotes proliferation by stimulating the ERK pathway. Wnt3a therefore stimulates the proliferation of fibroblast cells, at least in part, via activation of the ERK and Wnt/β-catenin pathways.

APC inhibits ERK pathway activation and cellular proliferation induced by RAS

Inactivating mutations in the adenomatous polyposis coli gene (APC), and activating mutations in RAS, occur in a majority of colorectal carcinomas. However, the relationship between these changes and tumorigenesis is poorly understood. RAS-induced activation of the ERK pathway was reduced by overexpressing APC in DLD-1 colorectal cancer cells. ERK activity was increased by Cre-virus-induced Apc knockout in primary Apcflox/flox mouse embryonic fibroblasts, indicating that APC inhibits ERK activity. ERK activity was increased by overexpression and decreased by knock down of β-catenin. The activation of Raf1, MEK and ERK kinases by β-catenin was reduced by co-expression of APC. These results indicate that APC inhibits the ERK pathway by an action on β-catenin. RAS-induced activation of the ERK pathway was reduced by the dominant negative form of TCF4, indicating that the ERK pathway regulation by APC/β-catenin signaling is, at least, partly caused by effects on β-catenin/TCF4-mediated gene expression. The GTP loading and the protein level of mutated RAS were decreased in cells with reduced ERK activity as a result of APC overexpression, indicating that APC regulates RAS-induced ERK activation at least partly by reduction of the RAS protein level. APC regulates cellular proliferation and transformation induced by activation of both RAS and β-catenin signaling.

p21Cip/WAF1 activation is an important factor for the ERK pathway dependent anti-proliferation of colorectal cancer cells

p21Cip/WAF1, an important regulator of cell proliferation, is induced by both p53- and extracellular signal regulated kinase (ERK) pathways. The induction of p21Cip/WAF1 occurs by prolonged activation of the ERKs caused by extracellular stimuli, such as zinc. However, not all the cells appeared to respond to ERK pathway dependent p21Cip/WAF1 induction. Here we investigated the cause of such difference using colorectal cancer cells. p21Cip/WAF1 induction and concomitant reduction of bromodeoxyuridine (BrdU) incorporation were observed by zinc treatment within HT-29 and DLD-1. However, HCT-116 cells with high endogenous p21Cip/WAF1 levels did not show any additional increment of p21Cip/WAF1 levels by zinc treatment and did maintain high BrdU incorporation level. The p21Cip/WAF1 induction by zinc depended upon prolonged activation of extracellular signal regulated kinase (ERK) was not observed in HCT-116 cells. The percentage of BrdU positive cells was 50% higher in p21Cip/WAF1 -/- HCT-116 cells compared to p21Cip/WAF1 +/+ HCT- 116 cells, and no cells induced p21Cip/WAF1 incorporated BrdU in its nucleus, yet confirming the importance of p21Cip/WAF1 induction in anti- proliferation. These results again support that p21Cip/WAF1 induction is a determinant in the regulation of colonic proliferation by the ERK pathway.

Akt is involved in the inhibition of cell proliferation by EGF

Axin is a negative regulator of the Wnt/β-catenin pathway and is involved in the regulation of axis formation and proliferation. Involvement of Axin in the regulation of other signaling pathways is poorly understood. In this study, we investigated the involvement of Akt in growth regulation by Axin in L929 fibroblasts stimulated by EGF. Akt activity was increased by EGF treatment and Ras activation, respectively. Both the EGF- and Ras-induced Akt activations were abolished by Axin induction, as revealed by both Western blot and immunocytochemical analyses. The proliferation and Akt activation induced by EGF were decreased by Axin induction, and the effects of EGF were abolished by treatment of an Akt-specific inhibitor. Therefore, Axin inhibits EGF-induced proliferation of L929 fibroblasts by blocking Akt activation.