Age-related changes in activation of mitogen-activated protein kinase cascades by oxidative stress.

Oxidative stress is thought to play a critical role in aging and the pathogenesis of human disease. Molecular studies of both the physiologic function of oxidants and the deleterious consequences of exposure to oxidative stress have suggested that signal transduction cascades may be targeted by oxidants. Here, we review recent studies from this laboratory examining the molecular basis for the activation of mitogen-activated protein kinases by oxidative stress and the influence of these pathways on cellular fate. We examine the association between constitutive activation of extracellular signal-regulated kinase (ERK) and cancer, and discuss how such mechanisms may contribute to oxidant-induced skin carcinogenesis. We also address the relationship between a decline in activation of this same pathway and the aged phenotype. In this regard, we review evidence that a decrease in activation of ERK by growth factor correlates with a reduced proliferative capacity in the isolated rat hepatocyte model, and we provide new data indicating that the activation of the ERK pathway in response to oxidant stimuli is also decreased with age. Further evidence demonstrates that this alteration is associated with both a reduced mitogenic response and a decline in hepatocyte cell survival in response to oxidative stress. Finally, we provide perspective on how modulations in ERK signaling may interplay with other changes in signal transduction cascades in the aging process.

Impairments in both p70 S6 kinase and extracellular signal-regulated kinase signaling pathways contribute to the decline in proliferative capacity of aged hepatocytes.

Treatment of primary cultured hepatocytes from adult (6-month-old) rats with epidermal growth factor (EGF) results in a marked elevation in DNA synthesis, a response that is markedly attenuated in cells of aged (24-month-old) animals. Recently we demonstrated that this age-related attenuation is associated with reduced activation of extracellular signal-regulated kinase (ERK) in response to EGF treatment. In order to gain further insight into the mechanisms responsible for the age-related decline in this proliferative response, we investigated the expression and/or activities of several other regulatory molecules important for G1 to S phase progression in EGF-stimulated young and aged hepatocytes. Induction of cyclin D1 and activation of cyclin-dependent kinase 2 (CDK2) by EGF were found to be diminished in the aged cells. In young cells, prior treatment with rapamycin inhibited the induction of DNA synthesis and activation of CDK2 to levels similar to those seen in aged cells without inhibiting ERK activity and cyclin D1 expression. This suggested that a distinct, ERK-independent, rapamycin-sensitive pathway might also contribute to the proliferative response in hepatocytes and be subject to age-related alterations. Further studies demonstrated that activation of p70 S6 kinase (p70S6k), a rapamycin-sensitive event, following EGF treatment was 40% lower in aged hepatocytes relative to young cells, although the kinetics of activation did not differ in the two age groups. Western blot analysis for p70S6k expression revealed similar levels of proteins in young and aged cells. From these findings, we conclude that deficiencies in both the ERK and p70S6k signaling pathways contribute to the age-related decline in the proliferative response of hepatocytes.

Aging reduces the numbers of hepatocytes synthesizing DNA in response to EGF and epinephrine.

Primary cultures of hepatocytes were prepared from young (6 month) and old (24 month) Wistar rats and exposed to epinephrine or epidermal growth factor. Incorporation of [3H]thymidine into DNA was determined both radiochemically and autoradiographically. The numbers of responding cells and degree of response per cell were determined and the results confirmed by FACScan analysis. Such analyses clearly demonstrate a reduced number of hepatocytes capable of responding to the above stimuli in cultures obtained from old rats. Thus, changes in numbers of responding cells may be an important mechanism involved in reduced responsiveness of the aged liver to agents which stimulate DNA synthesis and cell division.

Age-related decline in mitogen-activated protein kinase activity in epidermal growth factor-stimulated rat hepatocytes.

A number of studies have demonstrated that the proliferative capacity of cells declines with aging. In particular, epidermal growth factor (EGF)-stimulated DNA synthesis is reduced in hepatocytes from aged rats relative to young rats. Growth factor stimulation activates a genetic program in large part regulated by a family of mitogen-activated protein kinases (MAPK) that phosphorylate and thereby activate transcription factors involved in controlling the expression of proliferation-associated genes. In the present study, we compared the activation of the extracellular signal-regulated kinase 2 (ERK2) and c-Jun N-terminal kinase 1 (JNK1) MAPK in EGF-stimulated hepatocytes derived from young (6-month) and aged (24-month) rats. JNK activity was not appreciably altered by EGF treatment of cells from either age group. In contrast, ERK2 was highly activated by EGF treatment, but the magnitude of activation was significantly lower in hepatocytes of aged animals compared to those of young animals (7-fold versus 20-fold, respectively). The reduced ERK2 activity in response to EGF was associated with decreased c-fos and c-jun mRNA expression and lower levels of AP-1 transcription factor DNA binding activity in the aged hepatocytes. Finally, the basal expression of MAPK phosphatase 1, a MAPK-regulated gene involved in regulating MAPK activity, was higher in aged hepatocytes. Taken together, these findings suggest that an alteration in the balance between MAP kinase-phosphatase activities could contribute to the age-related decline in proliferative capacity.