Translational Regulation by P38 Mitogen–Activated Protein Kinase Signaling During Human Cholangiocarcinoma Growth

Abstract

The p38 mitogen-activated protein kinase (MAPK) signaling pathway is aberrantly expressed and maintains transformed cell growth in malignant human cholangiocytes. Because cell growth requires and is intimately related to protein synthesis, our aims were to assess the effect of p38 MAPK signaling on protein synthesis during growth of malignant human cholangiocytes. Inhibition of p38 MAPK activity during mitogenic stimulation decreased protein synthesis rates and tumor cell xenograft growth in nude mice. Altered protein synthesis resulted from decreased translational efficiency with impaired initiation of translation. Mitogenic stimulation resulted in phosphorylation of the eukaryotic initiation factor (eIF)-4E. Inhibition of p38 MAPK signaling or functional dysregulation of translation by small interfering double-stranded RNA (siRNA) to eIF-4E decreased anchorage-independent growth of malignant cholangiocytes. In conclusion, these studies identify a relationship between p38 MAPK activity and the regulation of protein synthesis during human cholangiocarcinoma growth. As protein synthesis is intimately linked to cell growth, dysregulation of translation initiation is a mechanism by which cellular p38 MAPK signaling participates in growth regulation of malignant cholangiocytes. (HEPATOLOGY 2003;38:158-166.)

Over-expression of interleukin-6 enhances cell survival and transformed cell growth in human malignant cholangiocytes

“…Moreover, inhibition of p38 MAPK activation decreases xenograft growth and anchorage independent growth of malignant cholangiocytes (24). These observations suggest that downstream targets regulated by p38 MAPK activation may contribute to promotion of tumor growth by IL-6.…”

“…To assess the effect of anchorage independent growth, cells (1,000 cells/35x10 mm plate) were grown in soft agar for 21 days at 37°C using a two layer agar system and the number of colonies quantitated as previously described (24).…”

“…KMCH cells were transfected as previously described (24). Briefly, 0.1 μg siRNA were mixed with 6 μl transfection agent (TransIt TKO, Mirus Corp., Madison, WI) and the mixture was incubated in 1 ml of media at room temperature for 15-20 min prior to adding to cultured cells grown to 50-60% confluency.…”

“…Similar results were observed in Mz-ChA-1, TFK-1 and CC-LP malignant human cholangiocytes after IL-6 treatment. Next, we assessed Mcl-1 expression using KM-p38dn cells, derived from KMCH malignant cholangiocytes stably transfected with a mutated MKK3 (A) that have reduced p38 MAPK activation following stimulation (24). In contrast to parental KMCH cells, IL-6 stimulation of KM-p38dn cells did not alter Mcl-1 expression.…”

“…Stable transfection was confirmed by reverse transcriptase PCR and using an IL-6 bioassay to verify IL-6 over-expression. For studies of p38 MAPK signaling pathways, KMCH-1 malignant human cholangiocytes cells were stably transfected with pRc/RSV-Flag MKK3(A), (encoding a dominant interfering upstream activator of p38 MAPK with double point mutations in Ser 189 and Thr 193 replaced by Ala) and designated as KM-p38dn, as previously described (24). Compared to control KM-1 cells, KM-p38dn cells have reduced activation of p38 MAPK.…”