Ganapathy K. Bhat

Caspases - an update.

Caspases belong to a family of highly conserved aspartate-specific cysteine proteases and are members of the interleukin-1beta-converting enzyme family, present in multicellular organisms. The caspase gene family consists of 15 mammalian members that are grouped into two major sub-families, namely inflammatory caspases and apoptotic caspases. The apoptotic caspases are further subdivided into two sub-groups, initiator caspases and executioner caspases. The caspases form a caspase-cascade system that plays the central role in the induction, transduction and amplification of intracellular apoptotic signals for cell fate determination, regulation of immunity, and cellular proliferation and differentiation. The substrates of apoptotic caspases have been associated with cellular dismantling, while inflammatory caspases mediate the proteolytic activation of inflammatory cytokines. The activation of this delicate caspase-cascade system and its functions are regulated by a variety of regulatory molecules, such as the inhibitor of apoptosis protein (IAP), FLICE, calpain, and Ca(2+). Based on the available literature we have reviewed and discussed the members of the caspase family, caspase-cascade system, caspase-regulating molecules and their apoptotic and non-apoptotic functions in cellular life and death. Also recent progress in the molecular structure and physiological role of non-mammalian caspases such as paracaspases, metacaspases and caspase-like-protease family members are included in relation to that of mammalian species.

Current concepts in apoptosis: the physiological suicide program revisited.

Apoptosis, or programmed cell death (PCD), involves a complex network of biochemical pathways that normally ensure a homeostatic balance between cellular proliferation and turnover in nearly all tissues. Apoptosis is essential for the body, as its deregulation can lead to several diseases. It plays a major role in a variety of physiological events, including embryonic development, tissue renewal, hormone-induced tissue atrophy, removal of inflammatory cells, and the evolution of granulation tissue into scar tissue. It also has an essential role in wound repair. The various cellular and biochemical mechanisms involved in apoptosis are not fully understood. However, there are two major pathways, the extrinsic pathway (receptor-mediated apoptotic pathway) and the intrinsic pathway (mitochondria-mediated apoptotic pathway), which are both well established. The key component in both is the activation of the caspase cascade. Caspases belong to the family of proteases that ultimately, by cleaving a set of proteins, cause disassembly of the cell. Although the caspase-mediated proteolytic cascade represents a central point in the apoptotic response, its initiation is tightly regulated by a variety of other factors. Among them, Bcl-2 family proteins, TNF and p53 play pivotal roles in the regulation of caspase activation and in the regulation of apoptosis. This review summarizes the established concepts in apoptosis as a physiological cell suicide program, highlighting the recent and significant advances in its study.

Folliculogenesis Is Impaired and Granulosa Cell Apoptosis Is Increased in Leptin-Deficient Mice

Abstract Leptin purportedly plays an important role in pubertal development in a number of mammalian species. Adult leptin-deficient (ob/ob) female mice are infertile, but the mechanisms responsible for the reproductive failure have not been fully elucidated. The major objective of the current study was to assess the effects of a leptin deficiency on ovarian folliculogenesis and apoptosis. Beginning at 4 wk of age, control (n = 8) and ob/ob (n = 7) mice were weighed and examined daily for vaginal opening. After 3 wk the mice were killed, and the reproductive organs were weighed. Ovaries were paraffin-embedded for hematoxylin and eosin histology, TUNEL assay, and immunohistochemistry for Fas, Fas ligand (FasL), and proliferating cell nuclear antigen (PCNA). Vaginal opening was delayed, uteri were smaller, and the number of primordial follicles and total number of ovarian follicles were subnormal in ob/ob animals. Leptin-deficient animals also had a higher number of atretic follicles than controls. Granulosa cells (predominantly in preantral and early antral follicles) of ob/ob mice exhibited increased apoptotic activity as documented by TUNEL assay and elevated expression of the apoptotic markers Fas and FasL, compared with that in control animals. Ovarian expression of PCNA, a marker of DNA replication, repair, or both, did not differ between ob/ob and control mice. The data suggest that a leptin deficiency in mice is associated with impaired folliculogenesis, which results in increased follicular atresia. This impairment may be one of the causative components of infertility in leptin-deficient animals.

Histone deacetylase inhibitors, valproic acid and trichostatin-A induce apoptosis and affect acetylation status of p53 in ERG-positive prostate cancer cells

An ETS family member, ETS Related Gene (ERG) is involved in the Ewing family of tumors as well as leukemias. Rearrangement of the ERG gene with the TMPRSS2 gene has been identified in the majority of prostate cancer patients. Additionally, overexpression of ERG is associated with unfavorable prognosis in prostate cancer patients similar to leukemia patients. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate transcription as well as epigenetic status of genes through acetylation of both histones and transcription factors. Deregulation of HATs and HDACs is frequently seen in various cancers, including prostate cancer. Many cellular oncogenes as well as tumor viral proteins are known to target either or both HATs and HDACs. Several studies have demonstrated that there are alterations of HDAC activity in prostate cancer cells. Recently, we found that ERG binds and inhibits HATs, which suggests that ERG is involved in deregulation of protein acetylation. Additionally, it has been shown that ERG is associated with a higher expression of HDACs. In this study, we tested the effect of the HDAC inhibitors valproic acid (VPA) and trichostatin-A (TSA) on ERG-positive prostate cancer cells (VCaP). We found that VPA and TSA induce apoptosis, upregulate p21/Waf1/CIP1, repress TMPRSS2-ERG expression and affect acetylation status of p53 in VCaP cells. These results suggest that HDAC inhibitors might restore HAT activity through two different ways: by inhibiting HDAC activity and by repressing HAT targeting oncoproteins such as ERG.

Mitochondrial localization, ELK-1 transcriptional regulation and growth inhibitory functions of BRCA1, BRCA1a, and BRCA1b proteins

BRCA1 is a tumor suppressor gene that is mutated in families with breast and ovarian cancer. Several BRCA1 splice variants are found in different tissues, but their subcellular localization and functions are poorly understood at the moment. We previously described BRCA1 splice variant BRCA1a to induce apoptosis and function as a tumor suppressor of triple negative breast, ovarian and prostate cancers. In this study we have analyzed the function of BRCA1 isoforms (BRCA1a and BRCA1b) and compared them to the wild‐type BRCA1 protein using several criteria like studying expression in normal and tumor cells by RNase protection assays, subcellular localization/fractionation by immunofluorescence microscopy and Western blot analysis, transcription regulation of biological relevant proteins and growth suppression in breast cancer cells. We are demonstrating for the first time that ectopically expressed GFP‐tagged BRCA1, BRCA1a, and BRCA1b proteins are localized to the mitochondria, repress ELK‐1 transcriptional activity and possess antiproliferative activity on breast cancer cells. These results suggest that the exon 9, 10, and 11 sequences (aa 263–1365) which contain two nuclear localization signals, p53, Rb, c‐Myc, γ‐tubulin, Stat, Rad51, Rad50 binding domains, angiopoietin‐1 repression domain are not absolutely required for mitochondrial localization and growth suppressor function of these proteins. Since mitochondrial dysfunction is a hallmark of cancer, we can speculate that the mitochondrial localization of BRCA1 proteins may be functionally significant in regulating both the mitochondrial DNA damage as well as apoptotic activity of BRCA1 proteins and mislocalization causes cancer. J. Cell. Physiol. 219: 634–641, 2009.

Impact of gonadotropin administration on folliculogenesis in prepubertal ob/ob mice

Female leptin deficient (ob/ob) mice exhibit abnormal ovarian folliculogenesis resulting in an impaired ability to reproduce. This effect may be related to the hypogonadotropic state of these animals, or leptin may directly modulate ovarian follicle development. In the present study we assessed whether exogenous gonadotropin administration would normalize folliculogenesis and induce ovulation in immature ob/ob animals. Eight 26-day-old ob/ob and eight control mice were injected sc with pregnant mare serum gonadotropin followed 48 h later with a sc injection of human chorionic gonadotropin. Animals were killed 24 h later. Gonadotropin (GTH) administration increased both ovarian and uterine weights in control mice, but this effect was attenuated in leptin deficient animals. The number of preantral follicles was greater in ob/ob mice than controls, but in GTH-treated animals the number of antral follicles was subnormal in the ovaries of leptin deficient animals. Ob/ob animals also failed to ovulate in response to GTH, and the protective actions of GTH against granulosa cell apoptosis and follicular atresia were attenuated in these animals. Interestingly, however, serum levels of estradiol and progesterone were higher in ob/ob mice than controls, regardless of whether or not the animals received GTH treatment. We conclude that the ovarian responsiveness to GTH is subnormal in leptin deficient animals suggesting that leptin may modulate the process of folliculogenesis by directly altering the sensitivity of the ovary to GTH.

Gonadotropin-Releasing Hormone-Agonist Inhibits Synthesis of Nitric Oxide and Steroidogenesis by Luteal Cells in the Pregnant Rat

Abstract We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo suppressed progesterone production and induced apoptosis in the corpus luteum (CL) of the pregnant rat. To investigate the mechanism(s) by which progesterone secretion is suppressed and apoptosis is induced in the luteal cells, we studied nitric oxide (NO) as a messenger molecule for GnRH action. Rats were treated individually on Day 8 of pregnancy with 5µg/day of GnRH-Ag for 4, 8, and 24 h. GnRH-Ag decreased the production of progesterone and pregnenolone 8 and 24 h after the administration. Corresponding with the reduction in these steroid hormones, luteal NO concentrations decreased at 8 and 24 h. Western blotting and immunohistochemical studies of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) in the CL demonstrated that administration of GnRH-Ag was associated with a marked decrease in eNOS and iNOS compared with sham controls at 4 and 8 h, but nNOS did not change throughout the experimental period. We demonstrated, for the first time, the presence of nNOS protein in the CL of the pregnant rat. To determine if this suppressive action of GnRH-Ag is directly on the CL, luteal cells were treated with GnRH-Ag for 4, 8, 12, and 24 h in vitro. Progesterone and NO concentrations in the media decreased at 8 and 12 h after the treatment and recovered at 24 h. Western blots revealed that eNOS and iNOS decreased in luteal cells treated with GnRH-Ag compared with controls at 4 and 8 h. These results demonstrate that suppression of luteal NO synthesis by GnRH-Ag is direct and leads to a decrease in the luteal production and release of progesterone and pregnenolone and thus suggest that GnRH could induce luteolysis in pregnant rats via NO.

Induction of a hypothyroid state during juvenile development delays pubertal reactivation of the neuroendocrine system governing luteinising hormone secretion in the male rhesus monkey (Macaca mulatta)

The present study aimed to determine the influence of thyroid status on the timing of the pubertal resurgence in gonadotrophin‐releasing hormone pulse generator activity [tracked by circulating luteinising hormone (LH) levels] in male rhesus monkeys. Six juvenile monkeys were orchidectomised and then treated with the antithyroid drug, methimazole, from 15–19 months until 36 months of age, at which time thyroxine (T4) replacement was initiated. Four additional agonadal monkeys served as controls. Blood samples were drawn weekly for hormonal assessments. Body weight, crown–rump length and bone age were monitored at regular intervals. By 8 weeks of methimazole treatment, plasma T4 had fallen sharply, and the decline was associated with a plasma thyroid‐stimulating hormone increase. In controls, plasma LH levels remained undetectable until the pubertal rise occurred at 29.3 ± 0.2 months of age. This developmental event occurred in only half of the methimazole‐treated animals before 36 months of age when T4 replacement was initiated. The hypothyroid state was associated with a profound arrest of growth and bone maturation, but increased body mass indices and plasma leptin levels. T4 replacement in methimazole‐treated monkeys was associated with the pubertal rise in LH in the remaining three animals and accelerated somatic development in all six animals. Although pubertal resurgence in LH secretion occurred at a later chronological age in methimazole‐treated animals compared to controls, bone age, crown–rump length and body weight at that time did not differ between groups. There were no long‐term differences in plasma prolactin between groups. We conclude that juvenile hypothyroidism in male primates causes a marked delay in the pubertal resurgence of LH secretion, probably occasioned at the hypothalamic level. Whether this effect is meditated by an action of thyroid hormone directly on the hypothalamus or indirectly as a result of the concomitant deficit in somatic development remains to be determined.

Clinostat Rotation Induces Apoptosis in Luteal Cells of the Pregnant Rat

Abstract Recent studies have shown that microgravity induces changes at the cellular level, including apoptosis. However, it is unknown whether microgravity affects luteal cell function. This study was performed to assess whether microgravity conditions generated by clinostat rotation induce apoptosis and affect steroidogenesis by luteal cells. Luteal cells isolated from the corpora lutea of Day 8 pregnant rats were placed in equal numbers in slide flasks (chamber slides). One slide flask was placed in the clinostat and the other served as a stationary control. At 48 h in the clinostat, whereas the levels of progesterone and total cellular protein decreased, the number of shrunken cells increased. To determine whether apoptosis occurred in shrunken cells, Comet and TUNEL assays were performed. At 48 h, the percentage of apoptotic cells in the clinostat increased compared with that in the control. To investigate how the microgravity conditions induce apoptosis, the active mitochondria in luteal cells were detected with JC-1 dye. Cells in the control consisted of many active mitochondria, which were evenly distributed throughout the cell. In contrast, cells in the clinostat displayed fewer active mitochondria, which were distributed either to the outer edge of the cell or around the nucleus. These results suggest that mitochondrial dysfunction induced by clinostat rotation could lead to apoptosis in luteal cells and suppression of progesterone production.

Does Leptin Mediate the Effect of Photoperiod on Immune Function in Mice

Abstract Seasonal fluctuations in immune status have been documented for avian and mammalian populations. During the late summer and early fall, immune function is bolstered to help animals cope with the more physiologically demanding winter. The environmental cue for these seasonal changes is apparently decreasing photoperiod. In the present study, we determined the potential role of leptin in mediating the effect of photoperiod on cell-mediated immune responses in male mice. Leptin-deficient (ob/ob) and littermate control mice were housed for 10 wk in either a short (8L:16D) or a long (16L:8D) photoperiod beginning at 6 wk of age. After the mice were killed, immune and reproductive organs were weighed and splenocytes isolated. The proliferative and cytokine responses (interleukin [IL]-2 and IL-4) of splenocytes to the T-cell mitogen, concanavalin A (Con A; 0–40 μg/ml), were determined. Body weights were elevated and both testes and seminal vesicle weights subnormal in ob/ob mice (by ANOVA, main effect of leptin deficiency), but thymuses and spleens were of normal size. Serum leptin levels were at minimum detection limits in ob/ob mice, but leptin levels in control mice housed at 8L:16D were higher than in control mice housed at 16L:8D. The proliferative response of splenocytes from ob/ob mice to Con A was subnormal (by ANOVA, main effect of leptin deficiency), but photoperiod had no effect on this response. Production of IL-2 in splenocytes of ob/ob mice was subnormal (by ANOVA, main effect of leptin deficiency) irrespective of photoperiod, but cells from mice housed at 8L:16D (by ANOVA, main effect of photoperiod) produced more IL-2 than cells from animals housed at 16L:8D. In contrast, a leptin deficiency did not alter IL-4 production, but cells from animals (ob/ob and controls) housed at 16L:8D produced less IL-4 than cells from animals housed at 8L:16D (by ANOVA, main effect of photoperiod). The present study suggests that both photoperiod and leptin have mutually independent effects on the proliferation of lymphocytes and cytokine production profiles. The data do not provide definitive support for the hypothesis that photoperiod-induced changes in leptin secretion mediate the effects of season on immune status.