Ravid Sasson

Cell-specific regulation of apoptosis by glucocorticoids: implication to their anti-inflammatory action.

Glucocorticoids play a major role in attenuation of the inflammatory response. These steroid hormones are able to induce apoptosis in cells of the hematopoietic system such as monocytes, macrophages, and T lymphocytes that are involved in the inflammation reaction. In contrast, it was discovered recently that in glandular cells such as the mammary gland epithelia, hepatocytes, ovarian follicular cells, and in fibroblasts glucocorticoids protect against apoptotic signals evoked by cytokines, cAMP, tumor suppressors, and death genes. The anti-apoptotic effect of glucocorticoids is exerted by modulation of several survival genes such as Bcl-2, Bcl-x(L), and NFkB, in a cell-specific manner. Moreover, upregulation or downregulation of the same gene product can occur in a cell-dependent manner following stimulation by glucocorticoids. This phenomenon is probably due to composite regulatory cross-talk among multiple nuclear coactivators or corepressors, which mediate the transcription regulation of the genes, by their interaction with the glucocorticoid receptor. These observations suggest that the anti-inflammatory action of glucocorticoids is exerted by two complementary mechanisms: on one hand, they induce death of the cells that provoke the inflammation, and on the other hand they protect the resident cells of the inflamed tissue by arresting apoptotic signals. Moreover, the complementary action of glucocorticoids provides a new insight to the therapeutic potential of these hormones.

The anti-inflammatory action of glucocorticoids is mediated by cell type specific regulation of apoptosis

Glucocorticoids play a major role in attenuation of the inflammatory response. These steroid hormones are able to induce apoptosis in cells of the hematopoietic system such as monocytes, macrophages and T-lymphocytes that are involved in the inflammation reaction. In contrast, it was discovered recently that in glandular cells such as the mammary gland epithelia, hepatocytes, ovarian follicular cells and in fibroblasts glucocorticoids protect against apoptotic signals evoked by cytokines, cAMP, tumor suppressors and death genes. The anti-apoptotic effect of glucocorticoids is exerted by modulation of several survival genes such as Bcl-2, Bcl-x(L) and NFkappaB, in a cell type-specific manner. Moreover, up regulation or down regulation of the same gene product can occur in a cell type-dependent manner following stimulation by glucocorticoids. This phenomenon is probably due to composite regulatory cross-talk among multiple nuclear coactivators or corepressors, which mediate the transcriptional regulation of the genes, by their interaction with the glucocorticoid receptor (GR). These observations suggest that the anti-inflammatory action of glucocorticoids is exerted by two complementary mechanisms: on the one hand, they induce death of the cells that provoke the inflammation, and on the other hand, they protect the resident cells of the inflamed tissue by arresting apoptotic signals.

Novel genes modulated by FSH in normal and immortalized FSH-responsive cells: new insights into the mechanism of FSH action

Follicle‐stimulating hormone (FSH) controls the development of follicle‐enclosed oocytes in the mammalian ovary by interacting with specific receptors located exclusively on granulosa cells. Its biological activity involves stimulation of intercellular communication, intracellular signaling, and up‐regulation of steroidogenesis; the entire spectrum of genes regulated by FSH is not yet fully characterized. We have established monoclonal rat FSH‐responsive granulosa cell lines that express FSH receptors at 20‐fold higher rates than with primary cells, and thus increased the probability of yielding a distinct spectrum of genes modulated by FSH. Using Affymetrix DNA microarrays, we discovered 11 genes not reported earlier to be up‐regulated by FSH and 9 genes not reported earlier to be down‐regulated by FSH. Modulation of signal transduction associated with G‐protein signaling, phosphorylation of proteins, and intracellular‐extracellular ion balance was suggested by up‐regulation of decay accelerating factor GPI‐form precursor (DAF), membrane interacting protein RGS16, protein tyrosine phosphatase (PTPase), oxidative stress‐inducible protein tyrosine phosphatase (OSIPTPase), and down‐regulation of rat prostatic acid phosphatase (rPAP), Na+, K+‐ATPase, and protein phosphatase 1β. Elevation in granzyme‐like proteins 1 and 3, and natural killer (NK) cell protease 1 (NKP‐1) along with reduction in carboxypeptidase E indicates possible FSH‐mediated preparation of the cells for apoptosis. Up‐regulation of vascular endothelial growth factors indicates the ability of FSH to produce angiogenic factors upon their maturation; whereas, reduction in insulin‐like growth factor binding protein (IGFBP3) indicates its increased potential to promote p53‐induced apoptosis. Striking similarities in FSH modulation of gene expression were found in primary cultures of human granulosa cells obtained from IVF patients although these cells expressed only 1% of FSH receptor compared with immortalized rat cells, as indicated by microarray technique, which probably is in the normal range of expression of this receptor in nontransformed cells. These findings should increase our understanding of the mechanism of FSH action in stimulating development of the ovarian follicular cells, of intracellular and intercellular communication, and of increasing the potential of ovarian follicular cells to undergo apoptosis during the process of selection of the dominant follicle.—OSasson, R., Dantes, A., Tajima, K., Amsterdam, A. Novel genes modulated by FSH in normal and immortalized FSH‐responsive cells: new insights into the mechanism of FSH action. FASEB J. 17, 1256–1266 (2003)

Steroidogenesis and apoptosis in the mammalian ovary

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrous/menstrual cycle by programmed cell death is essential to maintain the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, plays an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows systematic studies of the mechanisms that control steroidogenesis and apoptosis in granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated up to 24h following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or TNF-alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one not involving mitochondrial Cyt C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and affymetrix DNA microarray technology we discovered that granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells. Thus, the apoptotic signals could bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures cyclicity of estradiol and progesterone release in the estrous/menstruous cycle even during the initial stages of apoptosis.

Alternative pathways of ovarian apoptosis: death for life.

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrus/menstrual cycle by programmed cell death is essential for maintaining the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, play an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows for systematic studies of the mechanisms that control steroidogenesis and apoptosis of granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated for up to 24hr following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or tumor necrosis factor alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one that does not involve mitochondrial cytochrome C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and Affymetrix DNA microarray we discovered that Granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells, thereby allowing the apoptotic signals to bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures the cyclicity of estradiol and progesterone release in the estrus/menstrus cycle even during the initial stage of apoptosis.

Glucocorticoids protect against apoptosis induced by serum deprivation, cyclic adenosine 3',5'-monophosphate and p53 activation in immortalized human granulosa cells: involvement of Bcl-2.

Glucocorticoid hormones are known to enhance gonadotropin/cAMP-induced steroidogenesis in rat and human granulosa cells. As glucocorticoids induce apoptosis in numerous cell types, we investigated the role of glucocorticoids in the control of apoptosis in immortalized human granulosa cells (HO-23) transfected with a temperature-sensitive mutant of p53 (Val135). When HO-23 were incubated with forskolin in the presence or absence of dexamethasone (Dex) at 32 or 37 C, progesterone production was higher by 4- and 8-fold in the presence of Dex at 37 or 32 C, respectively (P < 0. 01). The expression of adrenodoxin (ADX), which is an intrinsic part of the cytochrome P450 side-chain cleavage enzyme system, remained the same in the presence or absence of Dex in forskolin-stimulated cells. Dex reduced apoptosis (to 33% of control) in cultures after activation of p53 by shifting the temperature from 37 to 32 C. Moreover, Dex suppressed apoptosis induced by serum deprivation (to 40% of control) or forskolin stimulati...

Crosstalk Among Multiple Signaling Pathways Controlling Ovarian Cell Death

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few but healthy embryos. Degeneration of the old corpora lutea in each estrous/menstrual cycle by programmed cell death is essential to maintain the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, plays an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows systematic studies of the mechanisms that control steroidogenesis and apoptosis of granulosa cells. These cells are the most abundant type of somatic follicular cell. Moreover, crosstalk between p53 and extracellular matrix components such as laminin, fibronectin and basic fibroblast growth factor, between cAMP- and p53-generated signals and between steroid hormones and Bcl-2, can explain some of the fine tuning that controls ovarian steroidogenesis and apoptosis. Further study of the mechanisms of ovarian cell death will lead to a better understanding of the processes involved and permit the formulation of novel strategies for the treatment of ovarian malfunctions, such as polycystic ovarian syndrome and ovarian cancer.

Induction of apoptosis in granulosa cells by TNFα and its attenuation by glucocorticoids involve modulation of Bcl-2

Tumor necrosis factor alpha (TNF alpha) plays a role in mammalian ovarian follicular development, steroidogenesis, ovulation, luteolysis, and atresia, but the exact mechanism of TNF alpha action is not completely understood. Induction of apoptosis and suppression of steroidogenesis by TNF alpha in primary preovulatory rat and human granulosa cells, as well as, in human granulosa cells immortalized by mutated p53, were characterized in the present work. Dexamethasone (Dex) and hydrocortisone efficiently suppressed TNF alpha-induced apoptosis in granulosa cells. TNF alpha dramatically reduced intracellular levels of Bcl-2, while Dex abrogated this reduction. TNF alpha reduced considerably intracellular levels of StAR protein, a key regulating factor in steroidogenesis. This reduction can be explained only in part by elimination of cells through apoptosis, since loss of steroidogenic capacity was much higher and faster than the rate and extent of loss of cell viability induced by TNF alpha, suggesting independent mechanisms for TNF alpha-induction of apoptosis and TNF alpha-suppression of steroidogenesis.

Pleiotropic anti-apoptotic activity of glucocorticoids in ovarian follicular cells.

Glucocorticoids (GC) such as hydrocortisone and dexamethasone (DEX) protect steroidogenic granulosa cells against apoptosis induced by serum deprivation, cAMP, tumor necrosis factor alpha stimulation or p53 activation. The protective effects were evident both in primary rat and human granulosa cells, which comprise the main population of the ovarian follicular cells, as well as in steroidogenic granulosa cell lines established in our laboratory. A correlation between the expression of Bcl-2 protein and protection against apoptosis induced by DEX was found in granulosa cell lines expressing various levels of Bcl-2. Incubation with DEX leads to development of a rigid network of actin cytoskeleton and increased incidence of adherence and gap junctions. Higher content of connexin 43 and total cadherins were found in GC stimulated cells compared to non-stimulated, suggesting that cell contact and intracellular communication contribute to the DEX induced resistance to apoptotic signals. Activation by DEX of MAPK and Akt/PKB but not p38 supported the view of a pleiotropic action of GC against apoptotic signals. Granzyme B, a protease characteristic for induction of apoptosis by T-cytotoxic lymphocytes and natural killer cells, was expressed and augmented during stimulation of apoptosis in the granulosa cells, and its synthesis and activation was blocked by DEX. It is concluded that GC exerted their anti-apoptotic effects in granulosa cells by multiple characteristic pathways. Moreover, the presence of endogenous granzyme B in granulosa cells suggest a novel intrinsic alternative apoptotic pathway that was earlier reported to be mediated uniquely by T-cytotoxic lymphocytes and natural killer cells. The anti-apoptotic effect of GC may play an important role in the healing process of the ovulatory follicle subsequent to follicular rupture and its rapid conversion to an active corpus luteum.

Generation and Application of Ovarian Steroidogenic Cell Lines

Primary granulosa cells of mammals such as rats, mice, and humans can be immortalized by transfecting them with oncogenes and mutated tumor suppressor genes. The established cell lines preserve their potential to undergo differentiation and luteinization under stimulation by substances elevating intracellular levels of cyclic adenosine monophosphate (cAMP). Moreover, if these cells are cotransfected with plasmids coding for the rat or human luteinizing hormone (LH), human chorionic gonadotropin (hCG), or follicle-stimulating hormone (FSH) receptors, they respond to gonadotropin stimulation by activation of hormone sensitive adenylate cyclase followed by de novo formation of steroid acute regulatory (StAR) protein; the cytochrome P450 side chain cleavage enzyme system; and subsequent formation of pregnenolone, progesterone, and 20a dihydroprogesterone. These cells also respond to gonadotropin/cAMP stimulation, elevating intracellular communication via gap junctions (GJs) and forming typical ovarian paracrine factors such as follistatin. This immortalized granulosa cell system is valuable for the study of mechanisms of apoptosis induced by serum deprivation, p53, and tumor necrosis factor-α (TNF-α), as well as the prevention of apoptosis by tyrosine kinase growth factors (e.g., basic fibroblast growth factor [bFGF]) and steroid hormones (e.g., glucocorticoids). Most recently, this cell system has become a useful tool to study genomic function modulated by gonadotropic hormones. The advantage of this cell system is its homogenous population of cells because each cell line is monoclonal. Thus endocrinological, biochemical, and molecular studies on the entire cell population can be applied to an individual cell type.