Sang Chul Park

Failure of stress-induced downregulation of Bcl-2 contributes to apoptosis resistance in senescent human diploid fibroblasts.

We previously reported that senescent human diploid fibroblasts (HDFs) are resistant to apoptosis induced by H2O2 and staurosporine. We report here that senescent HDFs are resistant to thapsigargin-induced apoptosis as well. These agonists caused the reductions in mitochondrial membrane potential (MMP) and in the apoptosis inhibitory protein (B-cell lymphoma) only in young HDFs but not in senescent HDFs. In addition, downregulation of Bcl-2 increased the sensitivity of senescent HDFs to apoptosis induction, suggesting the significant role of Bcl-2 in apoptosis resistance of the senescent HDFs. We further found that P-cAMP response element-binding protein (CREB), a positive regulator of Bcl-2, decreased in stress-induced apoptosis of young HDFs but not in senescent HDFs, and that Bcl-2 was markedly reduced in CREB small interfering RNA (siRNA), transfected senescent HDFs. In addition, activity of protein phosphatase 2A (PP2A), which dephosphorylates p-CREB, significantly increased in young HDFs but not in senescent HDFs treated with H2O2, staurosporine or thapsigargin. Taken together, these results suggest that failure of stress-induced downregulation of Bcl-2 underlies resistance of senescent HDFs to apoptosis.

Regulation of insulin response in skeletal muscle cell by caveolin status

Recent studies on the role of caveolin‐1 in adipocytes showed that caveolin has emerged as an important regulatory element in insulin signaling but little is known on its role in skeletal muscle cells. In this study, we demonstrate for the first time that caveolin‐1 plays a crucial role in insulin dependent glucose uptake in skeletal muscle cells. Differentiation of L6 skeletal muscle cells induce the expression of caveolin‐1 and caveolin‐3 with partial colocalization. However in contrast to adipocytes, phosphorylation of insulin receptor β (IRβ) and Akt/Erk was not affected by the respective downregulation of caveolin‐1 or caveolin‐3 in the muscle cells. Moreover, the phosphorylation of IRβ was detected not only in the caveolae but also in the non‐caveolae fractions of the muscle cells despite the interaction of IRβ with caveolin‐1 and caveolin‐3. These data implicate the lack of relationship between caveolins and IRβ pathway in the muscle cells, different from the adipocytes. However, glucose uptake was reduced specifically by downregulation of caveolin‐1, but not that of caveolin‐3. Taken together, these observations suggest that caveolin‐1 plays a crucial role in glucose uptake in differentiated muscle cells and that the regulation of caveolin‐1 expression may be an important mechanism for insulin sensitivity, implying the role of muscle cells for type 2 diabetes. J. Cell. Biochem. 99: 747–758, 2006.

Downstream molecular events in the altered profiles of lysophosphatidic acid-induced cAMP in senescent human diploid fibroblasts

Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ia. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.

Association between the MLH1 gene and longevity.

Perturbations in genomic stability result in cancer, a reduced life span, and premature aging. MLH1 is a mismatch repair enzyme that acts to maintain genomic stability, and a loss of MLH1 increases cancer incidence and apoptosis resistance, which suggests a link between MLH1 and longevity. We found here that MLH1 is associated with longevity by comparing a centenarian group with a control group. Our data indicate a critical role for MLH1 in longevity.

Exercise type and muscle fiber specific induction of caveolin-1 expression for insulin sensitivity of skeletal muscle

It is well known that exercise can have beneficial effects on insulin resistance by activation of glucose transporter. Following up our previous report that caveolin-1 plays an important role in glucose uptake in L6 skeletal muscle cells, we examined whether exercise alters the expression of caveolin-1, and whether exercise-caused changes are muscle fiber and exercise type specific. Fifity week-old Sprague Dawley (SD) rats were trained to climb a ladder and treadmill for 8 weeks and their soleus muscles (SOL) and extensor digitorum longus muscles (EDL) were removed after the last bout of exercise and compared with those from non-exercised animals. We found that the expression of insulin related proteins and caveolins did not change in SOL muscles after exercise. However, in EDL muscles, the expression of insulin receptor β (IRβ) and glucose transporter-4 (GLUT-4) as well as phosphorylation of AKT and AMPK increased with resistance exercise but not with aerobic exercise. Also, caveolin-1 and caveolin-3 increased along with insulin related proteins only in EDL muscles by resistance exercise. These results suggest that upregulation of caveolin-1 in the skeletal muscle is fiber specific and exercise type specific, implicating the requirement of the specific mode of exercise to improve insulin sensitivity.

Role of protein kinase C-dependent A-kinase anchoring proteins in lysophosphatidic acid-induced cAMP signaling in human diploid fibroblasts.

Previously, we reported that lysophosphatidic acid (LPA)‐induced adenosine 3′,5′‐cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A‐kinase anchoring proteins (AKAP) in the regulation of LPA‐stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)‐dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co‐immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium‐dependent PKCs α/β both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA‐dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Giα in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long‐term activation of PKC by LPA in senescent cells. We conclude that LPA‐dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium‐dependent PKC α/β and AC4/6.

Lysophosphatidic acid-induced changes in cAMP profiles in young and senescent human fibroblasts as a clue to the ageing process

This study attempts to elucidate the molecular mechanisms underlying the ageing-dependent cAMP profiles in human diploid fibroblasts stimulated by lysophosphatidic acid (LPA). In senescent cells, LPA-dependent Gialpha activation was reduced, with a consequent reduction in Gi-suppressed cAMP levels, without alterations in the levels of Gialpha proteins. In young cells, when Gialpha activity was inhibited by pertussis toxin pretreatment, or when its expression was blocked by siRNA, the pattern of changes in cAMP levels in response to LPA was similar to that seen in senescent cells. An increase in protein kinase C (PKC)-dependent isoforms of adenylyl cyclase (AC) types II, IV, and VI was also observed in these senescent fibroblasts. In senescent cells treated with PKC-specific inhibitors, bis-indolylmaleimide, Gö6976, rottlerin, and PKCvarepsilonV1, LPA-induced cAMP accumulation was inhibited, indicating that increased ACs in response to LPA occur via the activation of protein kinase Cs. When the expression of AC II, IV, and VI was blocked by siRNA in senescent fibroblasts, LPA-induced cAMP accumulation was also blocked. These results suggest that the senescence-associated increase of cAMP levels after LPA treatment is associated with reduced Gialpha, increased AC II, IV, and VI proteins, and PKC-dependent stimulation of their activities and provide an explanation for the age-dependent differences in cAMP-related physiological responses.

The seventh Korea–Japan joint symposium on cancer and ageing research: molecular targets in cancer and ageing research

There is a significant association between advanced age and an increased incidence of cancer. It is thus imperative to discuss issues related to cancer and ageing together. The unique symposium, the seventh Korea– Japan joint symposium on cancer and ageing research: molecular targets in cancer and ageing research, was held from 3 to 4 November 2005 at a nice resort town named Muju in Korea. This seventh symposium sponsored by the Journal of Cancer Research and Clinical Oncology brought together two groups of scientists working in the fields of cancer and ageing biology. This article summarizes the findings of this symposium, which highlights the intimate relationship between carcinogenesis and ageing. Plenary lectures, sessions, posters

Metabolic loading of guanosine induces chondrocyte apoptosis via the Fas pathway

Although the apoptosis of chondrocytes plays an important role in endochondral ossification, its mechanism has not been elucidated. In this study, we show that guanosine induces chondrocyte apoptosis based on the results of acridine orange/ ethidium bromide staining, caspase-3 activation, and sub-G1 fraction analysis. The potent inhibitory effect of dipyridamole, a nucleoside transporter blocker, indicates that extracellular guanosine must enter the chondrocytes to induce apoptosis. We found that guanosine promotes Fas-Fas ligand interaction which, in turn, leads to chondrocyte apoptosis. These findings indicate a novel mechanism for endochondral ossification via metabolic regulation.