Death receptors in cutaneous biology and disease.

Death receptors are a growing family of transmembrane proteins that can detect the presence of specific extracellular death signals and rapidly trigger cellular destruction by apoptosis. Expression and signaling by death receptors and their respective ligands is a tightly regulated process essential for key physiologic functions in a variety of organs, including the skin. Several death receptors and ligands, Fas and Fas ligand being the most important to date, are expressed in the skin and have proven to be essential in contributing to its functional integrity. Recent evidence has shown that Fas-induced keratinocyte apoptosis in response to ultraviolet light, prevents the accumulation of pro-carcinogenic p53 mutations by deleting ultraviolet-mutated keratinocytes. Further- more, there is strong evidence that dysregulation of Fas expression and/or signaling contributes to the pathogenesis of toxic epidermal necrolysis, acute cutaneous graft versus host disease, contact hypersensitivity and melanoma metastasis. With these new developments, strategies for modulating the function of death receptor signaling pathways have emerged and provided novel therapeutic possibilities. Specific blockade of Fas, for example with intravenous immunoglobulin preparations that contain specific anti-Fas antibodies, has shown great promise in the treatment of toxic epidermal necrolysis and may also be useful in the treatment acute graft versus host disease. Likewise, induction of death signaling by ultraviolet light can lead to hapten-specific tolerance, and gene transfer of Fas ligand to dendritic cells can be used to induce antigen specific tolerance by deleting antigen-specific T cells. Further developments in this field may have important clinical implications in cutaneous disease.

Clusterin gene expression mediates resistance to apoptotic cell death induced by heat shock and oxidative stress.

Clusterin is a widely expressed, well conserved, secreted glycoprotein, which is highly induced in tissues regressing as a consequence of apoptotic cell death in vivo. It has recently been shown that clusterin expression is only confined to surviving cells following the induction of apoptosis in vitro, suggesting that it is involved in cell survival rather than death. In the hypothesis that clusterin may be implicated in cellular responses to stress, clusterin gene expression was analyzed in the A431 human epidermoid cancer cell line following heat shock and oxidative stress. Our results show that both a transient heat shock (20 min at 42 degrees C) and various oxidative stresses, including hydrogen peroxide, superoxide anion, hyperoxia and UVA exposure, induce a strong increase in clusterin mRNA levels as assessed by northern blot. Nuclear run-on analysis suggests that transcriptional activation is involved in inducing clusterin mRNA in response to heat shock. Using pulse-chase analysis of control and heat shocked cells, it is shown that clusterin mRNA is translated and secreted, thus resulting in increased extracellular levels of the protein following heat shock. To investigate the function of clusterin in response to these stresses, clusterin anti-sense transfectants that stably express virtually no clusterin at the mRNA and protein level were generated in A431 cells. These anti-sense transfectants are shown to be highly sensitive to apoptotic cell death induced by heat shock or oxidative stress compared with wild-type A431 cells or control transfectants. Taken together, our results show that clusterin gene expression is induced in response to heat shock and oxidative stress in human A431 cells, and confers cellular protection against heat shock and oxidative stress.

Upregulation and redistribution of E-MAP-115 (epithelial microtubule-associated protein of 115 kDa) in terminally differentiating keratinocytes is coincident with the formation of intercellular contacts.

Microtubules are involved in the positioning and movement of organelles and vesicles and therefore play fundamental roles in cell polarization and differentiation. Their organization and properties are cell-type specific and are controlled by microtubule-associated proteins (MAP). E-MAP-115 (epithelial microtubule-associated protein of 115 kDa) has been identified as a microtubule-stabilizing protein predominantly expressed in epithelial cells. We have used human skin and primary keratinocytes as a model to assess a putative function of E-MAP-115 in stabilizing and reorganizing the microtubule network during epithelial cell differentiation. Immunolabeling of skin sections indicated that E-MAP-115 is predominantly expressed in the suprabasal layers of the normal epidermis and, in agreement with this observation, is relatively abundant in squamous cell carcinomas but barely detectable in basal cell carcinomas. In primary keratinocytes whose terminal differentiation was induced by increasing the Ca2+ concentration of the medium, E-MAP-115 expression significantly increased during the first day, as observed by northern and western blot analysis. Parallel immunofluorescence studies showed an early redistribution of E-MAP-115 from microtubules with a paranuclear localization to cortical microtubules organized in spike-like bundles facing intercellular contacts. This phenomenon is transient and can be reversed by Ca2+ depletion. Treatment of cells with cytoskeleton-active drugs after raising the Ca2+ concentration indicated that E-MAP-115 is associated with a subset of stable microtubules and that the cortical localization of these microtubules is dependent on other microtubules but not on strong interactions with the actin cytoskeleton or the plasma membrane. The mechanism whereby E-MAP-115 would redistribute to and stabilize cortical microtubules used for the polarized transport of vesicles towards the plasma membrane, where important reorganizations take place upon stratification, is discussed.

Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin.

Toxic epidermal necrolysis (TEN, Lyell's syndrome) is a severe adverse drug reaction in which keratinocytes die and large sections of epidermis separate from the dermis. Keratinocytes normally express the death receptor Fas (CD95); those from TEN patients were found to express lytically active Fas ligand (FasL). Antibodies present in pooled human intravenous immunoglobulins (IVIG) blocked Fas-mediated keratinocyte death in vitro. In a pilot study, 10 consecutive individuals with clinically and histologically confirmed TEN were treated with IVIG; disease progression was rapidly reversed and the outcome was favorable in all cases. Thus, Fas-FasL interactions are directly involved in the epidermal necrolysis of TEN, and IVIG may be an effective treatment.

Possible neuroprotective role of clusterin in Alzheimer's disease: a quantitative immunocytochemical study.

Clusterin is a secreted glycoprotein that is expressed in response to tissue injury both in peripheral organs and in the brain. Recent studies have shown a substantial increase in clusterin mRNA in pyramidal neurons of the hippocampus and the entorhinal cortex in Alzheimer's disease (AD), with clusterin immunoreactivity occurring in neuropil threads, neurofibrillary tangles (NFT), and senile plaques. To elucidate further the role of this protein in the degenerative process, a quantitative study of its distribution in the cerebral cortex of non-demented and AD patients, all older than 85 years of age, was performed using immunocytochemistry. Using a stereological approach, we found that in cortical areas affected in AD, such as the entorhinal, inferior temporal and superior frontal cortices, the percentage of NFT-free neurons displaying clusterin immunoreactivity was significantly higher than that in non-demented cases. No such increase in the density of clusterin-immunoreactive neurons was seen in cortical areas that were less affected in the disease process. Furthermore, clusterin immunoreactivity was rarely observed in NFT-containing neurons. In conjunction with previous observations in peripheral tissues, these data suggest that clusterin may have a neuroprotective role, and that in AD, low cellular expression of this protein may be associated with neuronal degeneration and death.

Fas ligand expression is restricted to nonlymphoid thymic components in situ.

The cell surface receptor Fas (Apo-1/CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in activation-induced death of mature T cells and in killing mediated by cytolytic T cells. The role of the Fas pathway in apoptosis associated with thymic selection events is, however, controversial. Although Fas and FasL are known to be expressed in the thymus, the nature and in vivo localization of FasL-expressing cells have not been determined. Using recently developed anti-FasL Abs in combination with in situ hybridization on tissue sections, we show in this work that FasL-expressing cells are present in the thymus, particularly within the medulla. FasL mRNA was detected readily in thymic stromal cell extracts, but not in isolated thymocytes. Moreover, immunohistochemical analysis of serial tissue sections stained with Abs against FasL in conjunction with epithelial and dendritic cell markers indicated that both thymic epithelial and dendritic cells express FasL in situ. The coexistence of FasL-expressing stromal cells and Fas-expressing thymocytes may have important implications for the role of the Fas pathway in apoptosis associated with thymic selection events.

Adrenocortical-specific transgene expression directed by steroid hydroxylase gene promoters.

The 5'-flanking regions of genes for three mouse adrenal steroid hydroxylases were analyzed for their ability to direct adrenal cortex-specific beta-galactosidase (beta-gal) reporter expression both in cell culture and transgenic mice. The 5'-flanking regions chosen were from the genes for steroid 21-hydroxylase (21-OHase), expressed throughout the adrenal cortex and mediating both glucocorticoid and mineralocorticoid synthesis, and aldosterone synthetase (AS) and steroid 11 beta-hydroxylase (11 beta-OHase), which catalyze respectively the terminal steps of mineralocorticoid synthesis in the zona glomerulosa and glucocorticoid synthesis in the zona fasciculata/reticularis. While 5.0 kb of 11 beta-OHase gene 5'-flanking region and 5.4 kb of the AS gene 5'-flanking region mediated respectively moderate and low levels of beta-gal reporter expression in Y1 adrenocortical tumor cells, neither of these 5'-flanking regions was able to direct reporter expression to the appropriate adrenocortical zone of transgenic mice. This suggests that additional regulatory elements, lying outside these 5'-flanking regions, are required for 11 beta-OHase and AS gene expression in the intact mouse. In contrast, 6.4 kb of the mouse 21-OHase A gene 5' flanking region was able to direct specific beta-galactosidase reporter expression, in both Y1 cells and transgenic mice, indicating that elements directing adrenal cortex-specific gene expression in vivo are located not more than 6.4 kb 5' of the 21-OHase gene transcription start site.

Variegated expression of a mouse steroid 21-hydroxylase/beta- galactosidase transgene suggests centripetal migration of adrenocortical cells.

5'-Flanking sequences (6.4 kb) of the mouse steroid 21-hyrodxylase (21-OHase) A gene linked to a LacZ reporter gene directed appropriate cell-specific expression in cultured Y1 adrenocortical tumor cells and in the adrenal cortex of transgenic mice. The transgene expression initiated at the same stage of adrenal development as the endogenous 21-OHase gene (embryonic day 11.5). Although the endogenous 21-OHase gene is expressed throughout the adrenal cortex, the 21-OHase/beta-gal transgene showed a strikingly variegated pattern of adrenocortical expression in all 10 transgene-expressing mouse lines examined. This presents as radial stripes of beta-gal staining transcending the classical zonal structure of the adrenal cortex but paralleling the columnar arrangement of cells of the zona fasciculata on the centripetal organization of the adrenocortical blood supply. To the extent that the variegated pattern of 21-OHase/beta-gal transgene expression depicts adrenocortical cell lineage, these results suggest that all cells within an individual stripe have a common clonal origin; the radial pattern of clonally derived cells argues that cellular migration maintains the adult adrenocortical cell population. Adrenal glands of developing embryos also exhibited a variegated pattern of 21-OHase/beta-gal transgene expression. However, this presented as islands of beta-gal reporter staining within the developing gland, suggesting that the rapid embryonic adrenal growth phase, which precedes the establishment of the classic adrenocortical zonal structure, may be governed by cellular mechanisms distinct from those responsible for maintenance of the adult adrenocortical cell population.

Regulation of primary response and specific genes in adrenal cells by peptide hormones and growth factors.

Using cultured bovine adrenal fasciculata cells (BAC), we investigated the effects of two hormones, corticotropin (ACTH) and angiotensin II (Ang-II) and two growth factors, insulin-like growth factors I (IGF-I) and transforming growth factor beta 1 (TGF beta 1), on the mRNA levels of nuclear proto-oncogenes of the Fos and Jun families and on the mRNA levels of genes expressed in BAC coding for ACTH and AT1 receptors, cytochrome P450scc and P450 17 alpha and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). ACTH and IGF-1 increased c-fos and jun-B mRNA levels early with later increases in the levels of mRNA for the ACTH receptor and the three steroidogenic enzymes, and enhanced steroidogenic responses to both ACTH and Ang-II. In contrast, Ang-II increased mRNA coding for the three proto-oncogenes (cfos, c-jun, and jun-B), decreased those for P450 17 alpha and 3 beta-HSD, and caused marked homologous and heterologous steroidogenic desensitization. TGF beta 1 increased only jun-B mRNA and markedly reduced BAC-differentiated functions and steroidogenic responsiveness to both ACTH and Ang-II. The long-term effects of ACTH on human adrenal fasciculata cells were comparable with those observed in BAC, whereas the long term effects of Ang-II and TGF beta 1 were different from those observed in BAC. Whether these species-specific differences are related to a different effect of these factors on proto-oncogene expression is not yet known.

Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover.

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. To gain further understanding of the Fas system., we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.

Adrenal-specific transgene expression and derivation of conditionally immortal rat adrenocortical cell lines.

Conditional immortalisation of cells is a powerful tool for establishing in vitro models maintaining a differentiated phenotype. We are utilising this approach to derive cell lines that maintain the characteristics of glomerulosa and fasciculata cells of the adrenal cortex. Such cell lines should provide a system in which to study aspects of adrenocortical function that are relevant to hypertension, such as the effects of the renin-angiotensin system on steroidogenesis.

Peptide hormone and growth factor regulation of nuclear proto-oncogenes and specific functions in adrenal cells.

Among the large number of immediate early genes, nuclear proto-oncogenes of the Fos and Jun families, have been postulated to be involved in the long-term effects of several growth factors on cell differentiation and/or multiplication. Since adrenal cell differentiated functions appear to be regulated by specific hormones and growth factors, the effects of these factors on proto-oncogene mRNA levels were analysed in bovine adrenal fasciculata cells (BAC) in culture. Corticotropin (ACTH) and insulin-like growth factor I increased c-fos and jun-B mRNA, but had no effect on c-jun mRNA and these early changes were associated with a later increase in BAC specific function [ACTH receptors, cytochrome P450 17 alpha) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)] and an enhanced steroidogenic responsiveness to both ACTH and angiotensin-II (A-II). On the other hand, A-II increased the three proto-oncogene (c-fos, c-jun and jun-B) mRNAs, induced a decrease of P450 17 alpha and 3 beta-HSD and caused a marked homologous and heterologous (ACTH) densitization. Transforming growth factor beta 1 which only increased jun-B mRNA, markedly reduced BAC differentiated functions and the steroidogenic responsiveness to both ACTH and A-II. Thus, it is postulated that the proto-oncoproteins encoded by the immediate early genes may play a role in the long-term effects of peptide hormones and growth factors on BAC differentiated functions.

Regulation of adrenal cell-differentiated functions by growth factors.

Adrenal cell-differentiated functions and, therefore, their steroidogenic capacity can be regulated in an opposite direction by insulin-like growth factor I (IGF-I) and transforming growth factor-beta 1 (TGF beta 1). The enhanced steroidogenic responsiveness of bovine and ovine adrenal cells treated with IGF-I can be explained by its positive effects on the number of corticotropin (ACTH) and angiotensin II (A-II) receptors associated with an increase in the alpha s and alpha i subunits of G proteins but also by its effects on several steps of the steroidogenic pathway. In contrast, TGF beta 1 is a potent inhibitor of differentiated functions of both bovine and ovine adrenal cells. TGF beta 1 reduces ACTH and A-II receptor number, inhibits cAMP formation, and decreases several steroidogenic enzyme activities. The physiological role of these peptides in adrenal cells is strengthened by the fact that both are synthetized and secreted by these cells and that their secretion can be regulated by the specific hormones, ACTH and A-II.

Regulation by growth factors (IGF-I, b-FGF and TGF-beta) of proto-oncogene mRNA, growth and differentiation of bovine adrenocortical fasciculata cells.

Nuclear proto-oncoproteins have been implicated in the regulation of gene expression by peptidic hormones and growth factors during cell proliferation and differentiation. In the present study we have investigated in bovine adrenal cells (BAC) the effects of basic fibroblast growth factor (b-FGF), insulin-like growth factor 1 (IGF-I) and transforming growth factor beta (TGF-beta) on c-jun, jun-B and c-fos mRNA levels and on cell growth and differentiation. Factors able to enhance the three proto-oncogenes (IGF-I, b-FGF and angiotensin II (A-II)) stimulate cell growth, whereas those inhibiting cell growth (TGF-beta and ACTH) decrease c-jun mRNA level. These results suggest that expression of c-jun may be required to induce cell proliferation. The relation between proto-oncogenes and the expression of differentiated functions appears to be more complex. Whereas IGF-I, b-FGF and A-II increase the three nuclear proto-oncogenes, the effects of IGF-I and b-FGF on cytochrome P450 17 alpha-hydroxylase mRNA levels are opposite to those of A-II. On the other hand, while TGF-beta and A-II have inhibitory effects on P450 17 alpha mRNA level, they have opposite effects on c-jun mRNA.

Angiotensin-II-induced expression of proto-oncogene (c-fos, jun-B and c-jun) mRNA in bovine adrenocortical fasciculata cells (BAC) is mediated by AT-1 receptors.

We have shown previously that angiotensin-II (A-II) controls proto-oncogene (c-fos, jun-B and c-jun) mRNA accumulation in bovine adrenal fasciculata cells (BAC). Since BAC contain both subtypes (AT-1 and AT-2) of the A-II receptor, we have investigated which subtype was involved in the effect of A-II on proto-oncogene mRNA by using a selective antagonist for AT-1 (DUP 753) and for AT-2 (CGP 42112A). DUP 753, but not CGP 42112A, inhibited the stimulatory effect of A-II on proto-oncogene mRNA, with ID50s of 4 x 10(-7) M, 7 x 10(-7) M and 2 x 10(-6) M for c-fos, jun-B and c-jun, respectively. Neither of the two antagonists by themselves had a direct effect on proto-oncogene mRNA. As the A-II AT-1 receptors are coupled to the phospholipase C system in BAC, we have investigated whether the A-II effects on the proto-oncogenes were mediated by protein kinase C (PKC) or by Ca2+ calmodulin. First, activation of PKC by the phorbol ester, PMA, increased the level of three proto-oncogene mRNAs, whereas calcium ionophore had no effect. Second, staurosporine, a specific inhibitor of PKC, reduced the stimulatory action of A-II on proto-oncogene mRNA by 80-90%, whereas trifluoroperazine, an inhibitor of calmodulin, had no significant effect. These results demonstrate that the effects of A-II on proto-oncogene mRNA are mediated by AT1 receptor subtypes, mainly through activation of the PKC pathway.

Regulation of c-fos, c-jun and jun-B messenger ribonucleic acids by angiotensin-II and corticotropin in ovine and bovine adrenocortical cells.

Previous work has shown that corticotropin (ACTH) and angiotensin-II (A-II), in addition to their acute steroidogenic effects, exert long-term influences on adrenal cell differentiated function, stimulatory or inhibitory, respectively. Certain nuclear proto-oncogenes have been implicated in the regulation of gene expression in many cell systems. We have investigated the effects of ACTH and A-II on the levels of c-fos, c-jun, and jun-B messenger RNAs (mRNAs), in bovine and ovine (OAC) adrenal fasciculata cells. In both cell types ACTH produced time- (maximum at 1 h) and dose-dependent (ED50 congruent to 10(-12) M) increase in c-fos (2- to 4-fold) and jun-B (10- to 20-fold) mRNA levels but did not affect c-jun. The concentrations required to induce half-maximal mRNA accumulation and cortisol production were similar. A-II also produced a dose-dependent increase in c-fos and jun-B mRNAs but also in c-jun in both cell types, despite the fact that OAC are resistant to the steroidogenic action of the hormone. The stimulatory effects of A-II on c-fos mRNA were higher than those produced by ACTH, whereas the effects on jun-B were similar but ACTH abolished (OAC) or decreased (bovine adrenal fasciculata cells) the stimulatory effects of A-II on c-jun mRNA. The effects of ACTH and A-II on cortisol production and proto-oncogene mRNAs were in part mimicked by 8 Bromo-cAMP and the phorbol ester phorbol-12-myristate-13 acetate plus calcium ionophore A23187, respectively. In the presence of cycloheximide, which blocks the steroidogenic effects of both hormones, proto-oncogene mRNAs were superinduced by both hormones. This result, together with the fact that dexamethasone failed to affect the mRNA levels suggests that the stimulatory effects of ACTH and A-II on proto-oncogene expression were not related to an autocrine/intracrine action of cortisol. Taken together, these findings show that the proto-oncogene mRNAs in normal adrenal cells are regulated by ACTH and A-II, acting through different intracellular pathways. They also demonstrate differential responsiveness of the Jun family to both hormones. Thus, the opposite long-term action of ACTH and A-II on adrenal cell differentiated function could be mediated by its different initial effects on proto-oncogene expression, in particular in the members of the Jun family.

Ovine adrenal fasciculata cells contain angiotensin-II receptors coupled to intracellular effectors but are resistant to the steroidogenic effects of this hormone.

Ovine adrenal fasciculata cells (OAC) responded to ACTH but were resistant to the steroidogenic action of angiotensin-II (A-II), while bovine adrenal fasciculata cells (BAC) responded to this hormone as well as to ACTH. However both cell types contained specific A-II binding sites (120,000 +/- 14,000 and 85,000 +/- 10,000 sites per cell for OAC and BAC, respectively) of similar high affinity [dissociation constant (KD) congruent to 2 x 10(-9) M]. Moreover, in both cell types, A-II receptors were coupled to intracellular effectors since A-II: 1) stimulated the accumulation of inositol phosphates, although the effects in BAC were higher than in OAC; 2) enhanced the influx and the efflux of 45Ca2+; 3) increased cytosolic free Ca2+ concentration ([Ca2+]i); 4) potentiated ACTH-induced cAMP production; and 5) induced A-II receptor loss. Both cell types appear to have an active protein kinase C since the phorbol ester 4 beta-phorbol 12-myristate-13-acetate potentiates ACTH-induced cAMP production and caused A-II receptor loss. In addition, 4 beta-phorbol 12-myristate-13-acetate and Ca2+ ionophore enhanced the steroid production by BAC but had no effect on OAC. These results indicated that the steroidogenic refractoriness of OAC to A-II might involve some step(s) beyond the initial activation of the two branches of the phosphoinositide pathway, activation of protein kinase C and increase of [Ca2+]i, and before conversion of cholesterol to pregnenolone.

Transforming growth factor beta treatment decreases ACTH receptors on ovine adrenocortical cells.

Transforming growth factor beta (TGF beta) is a potent inhibitor of adrenocortical cell differentiated functions, whereas corticotropin (ACTH) is the main physiological hormone which acts positively on these functions. We have studied the effects of both TGF beta and ACTH on ovine adrenocortical cell ACTH receptors. Ovine adrenocortical cells contained specific high affinity (Kd = 2.7 +/- 1.6 x 10(-10) M) and low capacity (1190 +/- 120 sites/cell) ACTH receptors. Pretreatment of cells with TGF beta resulted in a time- and dose-dependent (ED50 = 50 pg/ml) decrease of 125I-ACTH1-39 binding. The observed decrease in ACTH binding was due to a 2-3-fold decrease in the number of binding sites without modification of the binding affinity. On the contrary, pretreatment of cells with ACTH caused a 4-4.5-fold increase in the number of ACTH binding sites without an effect on the Kd. When cells were pretreated with both ACTH and TGF beta, TGF beta blocked completely the positive trophic effect of ACTH on its own receptors. The variations in ACTH receptor number were associated with parallel changes on acute ACTH-induced cyclic AMP production. Thus, the effects of TGF beta on ACTH receptor content are likely another important negative action of this peptide on adrenocortical cell differentiation. Moreover, these results suggest that regulation of ACTH receptor number may be one mechanism by which hormones and growth factors control adrenocortical differentiation.

Effects of transforming growth factor beta on ovine adrenocortical cells.

Transforming growth factor beta (TGF beta) is a potent regulator of steroidogenic cell function. However, the mechanisms of the effects are not well understood. We studied the actions of TGF beta on primary cultures of ovine adrenocortical (OAC) cells. OAC cells had high affinity receptors for TGF beta (KD congruent to 7.6 +/- 1.5 X 16(-11) M). In addition, TGF beta inhibited the following markers of adrenocortical function: (1) ACTH, cholera toxin and forskolin acute stimulation of cAMP and steroid production; (2) the acute 8-bromo-cAMP stimulation of corticosteroid and pregnenolone production; and (3) the activity and amount of P-450 17 alpha-hydroxylase protein as well as activities of 11 beta- and 21-hydroxylases. The inhibitory effects of TGF beta on ACTH-induced cAMP and steroid production were time (half inhibition at 6 and 3 h respectively) and dose dependent (ID50 congruent to 10(-12) M). From these data we concluded that TGF beta acted rapidly on sites of OAC cell acute responses to stimulation by ACTH before and after the production of cAMP. Pregnenolone production in these cells was not inhibited by TGF beta when steroid production was stimulated on the addition of the readily permeable cholesterol derivative, 22 R-hydroxycholesterol. Thus, the rapid effect on OAC cells was manifest by TGF beta action on the utilization of cellular pools of cholesterol for the acute stimulation of steroid formation and not by direct action on the cholesterol side-chain cleavage enzyme. In addition, cells stimulated with ACTH in the absence or presence of lipoproteins (for up to 36 h) were susceptible to the inhibitory action of TGF beta. Taken together, these data amplify the pleiotropic actions of TGF beta on adrenocortical cell function and demonstrate that one acute action of TGF beta is on the utilization of endogenous supplies of cholesterol for steroid production.

[Isolation of yeast protoplasts using various preparations of the hepato-pancreatic juice of Helix pomatia]. / Obtention de protoplastes de levure à l'aide de différences préparations de suc hépato-pancréatique d'Helix pomatia.

Conversion of large amounts of Saccharomyces cerevisiae cells to protoplasts is studied using various preparations extracted from Helix pomatia hepato-pancreatic juice. The most favourable yield in two hours incubations (88 per cent) is obtained with 20 ml cytohelicase, a chitinase and glucanase enriched extract, per 400 g of yeast cells, harvested at the end of the logarithmic growth phase and preincubated in presence of 2-mercaptoethanol.