John Papaconstantinou

Single-gene mutations that extend lifespan provide valuable tools for the exploration of the molecular basis for age-related changes in cell and tissue function and for the pathophysiology of age-dependent diseases. We show here that mice homozygous for loss-of-function mutations at the Pit1 (Snell dwarf) locus show a >40% increase in mean and maximal longevity on the relatively long-lived (C3H/HeJ x DW/J)F(1) background. Mutant dw(J)/dw animals show delays in age-dependent collagen cross-linking and in six age-sensitive indices of immune system status. These findings thus demonstrate that a single gene can control maximum lifespan and the timing of both cellular and extracellular senescence in a mammal. Pituitary transplantation into dwarf mice does not reverse the lifespan effect, suggesting that the effect is not due to lowered prolactin levels. In contrast, homozygosity for the Ghrhr(lit) mutation, which like the Pit1(dw) mutation lowers plasma growth hormone levels, does lead to a significant increase in longevity. Male Snell dwarf mice, unlike calorically restricted mice, become obese and exhibit proportionately high leptin levels in old age, showing that their exceptional longevity is not simply due to alterations in adiposity per se. Further studies of the Pit1(dw) mutant, and the closely related, long-lived Prop-1(df) (Ames dwarf) mutant, should provide new insights into the hormonal regulation of senescence, longevity, and late life disease.

Eucaryotic organisms possess natural defense processes triggered by stress factors such as injury, infection, and inflammation. The acute phase response is an early defense mechanism during which striking changes in protein synthesis occur in the liver and other tissues. The altered expression of many acute phase protein genes is at the transcriptional level. Some of these genes have DNA binding sites for the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. We report here that in vivo expression of three isoforms of C/EBP is dramatically changed during the acute phase response. The steady-state mRNA levels of C/EBP alpha decreased significantly in the liver, lung, and fat tissues of lipopolysaccharide (LPS)-treated mice; moreover, nuclear run-off transcription assays indicated a decrease in the rate of C/EBP alpha gene transcription in isolated liver nuclei. The steady-state levels of C/EBP beta and a new isoform, C/EBP delta, were dramatically increased in many tissues within 4 h following LPS treatment. The rates of transcription of these two genes were only minimally altered in liver but significantly increased in kidney nuclei isolated from stimulated animals. Thus, the C/EBP isoforms exhibit differential mechanisms in their responses to LPS in various tissues and are likely to play an important role in mediating the transcriptional activation of genes involved in the acute phase response.

I have presented a series of observations on macromolecular interactions which occur during the terminal stages of lens cell differentiation. These are summarized in Fig. 2. Other cell types that undergo similar changes are the erythrocyte and skin cells (epidermis) during the process of keratinization. These other cells are also involved in the synthesis of highly specific proteins, and there are indications that molecular alterations similar to those described for the lens may also occur in these cells (26). Thus, elucidation of a specific series of macromolecular initeractions such as those described may provide a basis for the biochemical definition of the terminal stages of cellular differentiation. Differentiation of the reticulocyte, for example, involves inactivation of the nucleus, stabilization of mRNA, and possibly a ribosomal breakdown such as I have described here (26). Furthermore, elucidation of the mechanisms of reactions involving the initiation of tissue-specific protein synthesis and suLbsequent nuclear inactivation, stabilization of mRNA, and breakdown of the ribosomes may provide a basis for defining the mechanisms of terminal cellular differentiation. The lens cell has reached its highest form of cellular differentiation when it has formed the fiber cell. With respect to the mechanism of lens fiber cell formation, we would like to know whether specific biochemical changes such as gamma-crystallin synthesis are intiniately linked to fiber cell formation-that is, whether gamma-crystallins are required to bring about the formation of a fiber cell. The potential for synthesizing gamma-crystallins is inherent in the genome of the cell. This part of the genome is nonfunctional in the epithelial cell. Can these genes be activated without bringing about a simultaneous cellular elongation, nuclear inactivation and loss of cellular replication, stabilization of mRNA, and breakdown of the ribosomes? The degree of coupling or uncoupling of tissue-specific-protein synthesis to morphogenesis is an important part of the mechanism of cellular differentiation. We feel that we have now reached the stage where we can begin to answer these questions.

The ASK1-signalosome→p38 MAPK and SAPK/JNK signaling networks promote senescence (in vitro) and aging (in vivo, animal models and human cohorts) in response to oxidative stress and inflammation. These networks contribute to the promotion of age-associated cardiovascular diseases of oxidative stress and inflammation. Furthermore, their inhibition delays the onset of these cardiovascular diseases as well as senescence and aging. In this review we focus on whether the (a) ASK1-signalosome, a major center of distribution of reactive oxygen species (ROS)-mediated stress signals, plays a role in the promotion of cardiovascular diseases of oxidative stress and inflammation; (b) The ASK1-signalosome links ROS signals generated by dysfunctional mitochondrial electron transport chain complexes to the p38 MAPK stress response pathway; (c) the pathway contributes to the sensitivity and vulnerability of aged tissues to diseases of oxidative stress; and (d) the importance of inhibitors of these pathways to the development of cardioprotection and pharmaceutical interventions. We propose that the ASK1-signalosome regulates the progression of cardiovascular diseases. The resultant attenuation of the physiological characteristics of cardiomyopathies and aging by inhibition of the ASK1-signalosome network lends support to this conclusion. Importantly the ROS-mediated activation of the ASK1-signalosome p38 MAPK pathway suggests it is a major center of dissemination of the ROS signals that promote senescence, aging and cardiovascular diseases. Pharmacological intervention is, therefore, feasible through the continued identification of potent, non-toxic small molecule inhibitors of either ASK1 or p38 MAPK activity. This is a fruitful future approach to the attenuation of physiological aspects of mammalian cardiomyopathies and aging.