Gertrude C. Kokkonen

Restraint of Proinflammatory Cytokine Biosynthesis by Mitogen-Activated Protein Kinase Phosphatase-1 in Lipopolysaccharide-Stimulated Macrophages

Exposure of macrophages to LPS elicits the production of proinflammatory cytokines, such as TNF-α, through complex signaling mechanisms. Mitogen-activated protein (MAP) kinases play a critical role in this process. In the present study, we have addressed the role of MAP kinase phosphatase-1 (MKP-1) in regulating proinflammatory cytokine production using RAW264.7 macrophages. Analysis of MAP kinase activity revealed a transient activation of c-Jun N-terminal kinase (JNK) and p38 after LPS stimulation. Interestingly, MKP-1 was induced concurrently with the inactivation of JNK and p38, whereas blocking MKP-1 induction by triptolide prevented this inactivation. Ectopic expression of MKP-1 accelerated JNK and p38 inactivation and substantially inhibited the production of TNF-α and IL-6. Induction of MKP-1 by LPS was found to be extracellular signal-regulated kinase dependent and involved enhanced gene expression and increased protein stability. Finally, MKP-1 expression was also induced by glucocorticoids as well as cholera toxin B subunit, an agent capable of preventing autoimmune diseases in animal models. These findings highlight MKP-1 as a critical negative regulator of the macrophage inflammatory response, underscoring its premise as a potential target for developing novel anti-inflammatory drugs.

Loss in oxidative stress tolerance with aging linked to reduced extracellular signal-regulated kinase and Akt kinase activities

Oxidative stress is believed to be an important factor in the development of age‐related diseases, and studies in lower organisms have established links between oxidative stress tolerance and longevity. We have hypothesized that aging is associated with a reduced ability to mount acute host defenses to oxidant injury, which increases the vulnerability of aged cells to stress. We tested this hypothesis by using primary hepatocytes from young (4‐6 months) and aged (24‐26 months) rats. Old hepatocytes were more sensitive to H2O2‐induced apoptosis than were young cells. Lower survival was associated with reduced activations of extracellular signal‐regulated kinase (ERK) and Akt kinase, both of which protect against oxidant injury. That reduced ERK and Akt activities contribute to lower survival of aged cells was supported by additional findings. First, pharmacologic inhibition of ERK and Akt activation in young cells markedly increased their sensitivity to H2O2. Second, caloric restriction, which increases rodent life span and delays the onset of many age‐related declines in physiologic function, prevented loss in ERK and Akt activation by H2O2 and enhanced survival of old hepatocytes to levels similar to those of young cells. Strategies aimed at boosting these host responses to acute oxidant injury could have significant anti‐aging benefits.

D2 Dopamine Receptors Stimulate Mitogenesis Through Pertussis Toxin-Sensitive G Proteins and Ras-Involved ERK and SAP/JNK Pathways in Rat C6-D2L Glioma Cells

Abstract: Dopamine D2 receptors are members of the G protein‐coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal‐regulated kinase (ERK) and c‐Jun NH2‐terminal kinase (JNK) pathways through a mechanism involving D2 receptor‐G protein complexes and the Ras GTP‐binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA‐induced kinase activation was prevented by either (+)‐butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (−)‐butaclamol, the inactive isomer of (+)‐butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA‐induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1(K → R) vector, a dominant negative SEK1 mutant, specifically prevented DA‐induced JNK activation and subsequent c‐Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [3H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras‐dependent mitogen‐activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA‐induced DNA synthesis. Transfection with FlagΔ169, a dominant negative c‐Jun mutant, also prevented stimulation of [3H]thymidine incorporation by DA. The demonstration of D2 receptor‐stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.

Caveolin-Induced Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway Increases Arsenite Cytotoxicity

ABSTRACT The inhibitory effect of caveolin on the cellular response to growth factor stimulation is well established. Given the significant overlap in signaling pathways involved in regulating cell proliferation and stress responsiveness, we hypothesized that caveolin would also affect a cells ability to respond to environmental stress. Here we investigated the ability of caveolin-1 to modulate the cellular response to sodium arsenite and thereby alter survival of the human cell lines 293 and HeLa. Cells stably transfected with caveolin-1 were found to be much more sensitive to the toxic effects of sodium arsenite than either untransfected parental cells or parental cells transfected with an empty vector. Unexpectedly, the caveolin-overexpressing cells also exhibited a significant activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which additional studies suggested was likely due to decreased neutral sphingomyelinase activity and ceramide synthesis. In contrast to its extensively documented antiapoptotic influence, the elevated activity of Akt appears to be important in sensitizing caveolin-expressing cells to arsenite-induced toxicity, as both pretreatment of cells with the PI3K inhibitor wortmannin and overexpression of a dominant-negative Akt mutant markedly improved the survival of arsenite-treated cells. This death-promoting influence of the PI3K/Akt pathway in caveolin-overexpressing cells appeared not to be unique to sodium arsenite, as wortmannin pretreatment also resulted in increased survival in the presence of H2O2. In summary, our results indicate that caveolin-induced upregulation of the PI3K/Akt signaling pathway, which appears to be a death signal in the presence of arsenite and H2O2, sensitizes cells to environmental stress.

Identification of neuronal programmed cell death in situ in the striatum of normal adult rat brain and its relationship to neuronal death during aging

Apoptotic neurons have been identified in normal adult rat striatum by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling technique. This observation suggests that neuronal programmed cell death starts at an early stage of adult life and may contribute to the aging associated neuronal loss. In addition, the frequency of apoptotic cells was found to significantly increase in old rats, which implies that aging itself accelerates the process.

Relationship between Nutrition and Aging

One of the most critical needs in nutritional research at the present time is a knowledge of the levels of intake of various nutrients at specific intervals in the life cycle which will optimize physical and mental development, physiological performance during adulthood, and the retention of health and vigor in senescence. In the past, emphasis had been placed primarily on the establishment of the nutritional requirements of young growing animals. Few efforts have been made to determine whether changes occur in nutritional requirements following growth cessation. This is unfortunate because many age changes in physiological functions may result in increased nutritional needs in later life. In addition, it is generally accepted that intakes moderately above the recommended allowances are optimal for the well-being of an organism. However, a number of studies carried out on animals have demonstrated that longevity was increased when intakes of certain nutrients were lower than the recommended allowances. Therefore, an attempt is made here to review the pertinent literature on (1) the effect of age on nutritional requirements following the cessation of growth and (2) the effect of nutrition on lifespan. This information may provide useful knowledge for the optimal nutrition of the aged and for an understanding of the basic mechanisms of biological aging. Studies concerned with the first problem area heve been carried out principally in human subjects, whereas those with the second area, in animals.

Diet and life extension in animal model systems.

Recent studies have shown that beneficial effects can be brought about when underfeeding is initiated in adult as well as young growing animals. In addition, such dietary manipulations have been shown to delay the onset of a variety of diseases although its relationship to total incidence has not been established. It has been proposed that dietary restriction reduces protein synthesis and increases lifespan by retarding genetic informational transfer during early life and reducing the use of the genetic code and thereby minimizing genetic imperfections as they may occur during late life.

Effect of aging on populations of estrogen receptor-containing cells in the rat uterus

Estrogen receptor-containing cells were identified in uteri of mature and senescent rats by means of quantitative autoradiography. Cell numbers and density were assessed in the various uterine layers. It was determined that despite decreases in receptor concentrations in endometrium, endometrial stroma, myometrium, and epithelium, no significant cell loss occurred in any region. Receptor content per cell clearly decreased with age in the endometrial stroma, while only in the myometrium was there any suggestion of cell loss in the absence of receptor loss from individual cells. Thus, age-related reductions in uterine estradiol receptor levels may be due to different mechanisms in different tissue regions.

Effects of aging and calorie restriction of Fischer 344 rats on hepatocellular response to proliferative signals

It is well established that the proliferative potential of the liver declines with aging. Epidermal growth factor (EGF)-stimulated DNA synthesis is reduced in hepatocytes from aged rats relative to young rats, and this reduction correlates with diminished activation of the extracellular signal-regulated kinase (ERK) pathway and lower phosphorylation of the EGF receptor on residue Y1173. Calorie restriction (CR) can increase rodent life span and retard many age-associated declines in physiologic function, but its influence on cell proliferation is unknown. Here, we investigated the effects of long-term CR on proliferation of hepatocytes derived from young and aged rats following in vitro stimulation with either low-dose hydrogen peroxide or EGF. CR reduced the proliferative response of hepatocytes derived from young hosts, but long-term CR was associated with enhanced proliferation in aged cells relative to that of ad libitum (AL)-fed animals. ERK activation mirrored the effects of CR on proliferation, in that young CR cells exhibited lower ERK activation than young AL cells, but old CR cells showed higher ERK activation than old AL cells. Finally, a decline in EGF receptor phosphorylation on Y1173, which normally occurs with aging, was absent in cells of old hosts maintained on long-term CR, supporting the view that alterations in this early signaling event underlie the age-related decline in proliferative potential in rat hepatocytes.

Dopamine stimulates redox-tyrosine kinase signaling and p38 MAPK in activation of astrocytic C6-D2l cells

An increase in dopamine (DA) availability in rat brain has been suggested to participate in certain neurodegenerative processes. However, the regulatory effects of DA on glial cells have not been extensively studied. Using a rat C6 glioma cell line stably expressing recombinant D2L receptors, we have found that micromolar levels of DA stimulate mitogenesis and glial fibrillary acidic protein (GFAP) expression, both serving as parameters of reactive gliosis. This mitogenesis occurs about 29 h after exposure to DA and requires D2-receptor-mediated intracellular redox-tyrosine kinase activation. Either DA or quinpirole, a D2 receptor agonist, stimulates protein tyrosine phosphorylation. Application of either DPI, a potent inhibitor of NADPH-dependent oxidase, or NAC, an anti-oxidant, effectively prevented DA-induced tyrosine phosphorylation and DNA synthesis. Preincubation of (+)-butaclamol, a D2 receptor antagonist, inhibits both DA-stimulated tyrosine phosphorylation and mitogenesis. DA at micromolar levels also stimulates GFAP expression. This DA-regulated GFAP expression can be completely inhibited by SB203580, a selective p38 MAPK inhibitor, but not influenced by (+)-butaclamol and genistein, a protein tyrosine kinase inhibitor. Thus, our data suggest that regulation of DNA synthesis and GFAP expression induced by DA is mediated by independent signaling pathways. The mitogenesis requires a D2-receptor-mediated protein tyrosine kinase cascade, while GFAP expression needs a D2-receptor-independent p38 MAPK activation. This observation may help to understand the processes of reactive gliosis in some dopaminergic-related neurodegenerative diseases.