Functional studies of MAPK signaling in regulating autophagy in renal cells and proliferation in embryonic stem cells

Abstract

Kidney disease is a serious health problem worldwide, which significantly lowers a person’s quality of life. Autophagy, an evolutionarily conserved lysosomal degradation pathway, is known to regulate many important physiological and pathological processes that play critical roles in health and disease. At present, the involvement of autophagy in kidney function and the precise mechanism of autophagy regulation in kidney remain elusive. In the current study, we aim to explore the role of ERK1 and ERK2 (referred as ERK1/2 hereafter), two related serine/threonine protein kinases in the RAF-MEK-ERK signaling cascade, in autophagy regulation in renal tubule epithelial cells under glucose starvation. ERK1/2 are the best studied mitogen-activated protein kinases (MAPKs) and they play an important role in a wide variety of cellular processes. Our preliminary data demonstrate that glucose starvation potently activated ERK1/2 in renal tubule epithelial cells, and this was concomitant with an increase in autophagic flux. Perturbing ERK1/2 activation by treatment with inhibitors of RAF; or via the expression of a dominant negative mutant form of MEK1/2; or via knockdown of RAF, blocked the glucose starvation-mediated ERK1/2 activation and induction of autophagy in renal tubule epithelial cells, rendering these cells more vulnerable to cell death. Moreover, we immuno-purified the ERK1/2 complex in renal tubule epithelial cells and found that glucose starvation induced an association between the members of the ULK1-ATG13-FIP200 complex, which is important for autophagy induction, and ERK1/2. We hypothesize that glucose starvation activates ERK1/2 signaling to positively regulate the autophagy machinery and protect the renal cells from damage, which will increase our understanding of autophagy regulation in the kidney, provide novel biomarkers for renal disease diagnosis, and promote the application of autophagy-based approaches in the treatment and prevention of human renal disease. ERK1/2 have been indicated to be dispensable for self-renewal of mouse ES cells. Simultaneously suppressing both ERK and GSK signaling not only allows self-renewal of ES cells independent of extracellular stimuli, but also enables more efficient derivation of naïve ES cells from rat and mouse strains. Interestingly, under the regular medium condition containing LIF and BMP, ERK1/2 are activated. However, whether the upstream factors of ERK1/2 are involved in priming or promoting ES cells differentiation has not been examined. Here we found that RAF family members, including A-RAF, B-RAF and C-RAF, are expressed in mouse ES cells. Single RAF knockdown did not change basal activity of ERK1/2, neither did A-RAF and B-RAF double knockdown nor B-RAF and C-RAF double knockdown change it in ES cells. Interestingly, B/C-RAF double knockdown, not A/B-RAF double knockdown, inhibited LIF-induced maximal ERK1/2 activation, accompanying with slower growth of ES cells. Moreover, A-RAF, B-RAF and C-RAF triple knockdown dramatically attenuated LIF-induced sustained and maximal ERK activity in ES cells. RAF triple knockdown, similar as treatment with U0126 (a MEK specific inhibitor), markedly decreased the proliferation and cell survival of ES cells, thereby compromising the colony propagation of ES cells. Collectively, our work indicate that RAFs-ERK1/2 cascade is required for growth and cell survival of mouse ES cells.