Biomedical subjects

M E Reyland

Publications and source records attributed to M E Reyland.

At least 19 recordsLinked to original sources

PubMed2003

Inhibition of PKCalpha induces a PKCdelta-dependent apoptotic program in salivary epithelial cells.

We have used expression of a kinase dead mutant of PKCalpha (PKCalphaKD) to explore the role of this isoform in salivary epithelial cell apoptosis. Expression of PKCalphaKD by adenovirus-mediated transduction results in a dose-dependent induction of apoptosis in salivary epithelial cells as measured by the accumulation of sub-G1 DNA, activation of caspase-3, and cleavage of PKCdelta and PKCzeta, known caspase substrates. Induction of apoptosis is accompanied by nine-fold activation of c-Jun-N-terminal kinase, and an approximately two to three-fold increase in activated mitogen-activated protein kinase (MAPK) as well as total MAPK protein. Previous studies from our laboratory have shown that PKCdelta activity is essential for the apoptotic response of salivary epithelial cells to a variety of cell toxins. To explore the contribution of PKCdelta to PKCalphaKD-induced apoptosis, salivary epithelial cells were cotransduced with PKCalphaKD and PKCdeltaKD expression vectors. Inhibition of endogenous PKCdelta blocked the ability of PKCalphaKD to induce apoptosis as indicated by cell morphology, DNA fragmentation, and caspase-3 activation, indicating that PKCdelta activity is required for the apoptotic program induced under conditions where PKCalpha is inhibited. These findings indicate that PKCalpha functions as a survival factor in salivary epithelial cells, while PKCdelta functions to regulate entry into the apoptotic pathway.

Adenoviridae↗

PubMed2001

PKCdelta is required for mitochondrial-dependent apoptosis in salivary epithelial cells.

We report here that the novel protein kinase C isoform, PKCdelta, is required at or prior to the level of the mitochondria for apoptosis induced by a diverse group of cell toxins. We have used adenoviral expression of a kinase-dead (KD) mutant of PKCdelta to explore the requirement for PKCdelta in the mitochondrial-dependent apoptotic pathway. Expression of PKCdeltaKD, but not PKCalphaKD, in salivary epithelial cells resulted in a dose-dependent inhibition of apoptosis induced by etoposide, UV-irradiation, brefeldin A, and paclitaxel. DNA fragmentation was blocked up to 71% in parotid C5 cells infected with the PKCdeltaKD adenovirus, whereas caspase-3 activity was inhibited up to 65%. The activation of caspase-9-like proteases by all agents was also inhibited in parotid C5 cells expressing PKCdeltaKD. The ability of PKCdeltaKD to block the loss of mitochondrial membrane potential was similarly determined. Expression of PKCdeltaKD blocked the decrease in mitochondrial membrane potential observed in cells treated with etoposide, UV, brefeldin A, or paclitaxel in a dose-dependent manner. In contrast to the protective function of PKCdeltaKD, expression of PKCdeltaWT resulted in a potent induction of apoptosis, which could be inhibited by co-infection with PKCdeltaKD. These results suggest that PKCdelta is a common intermediate in mitochondrial-dependent apoptosis in salivary epithelial cells.

Adenoviridae↗

PubMed2001

Neuroprotection of grafted neurons with a GDNF/caspase inhibitor cocktail.

Transplantation of fetal ventral mesencephalic (VM) tissue shows great promise as an experimental therapy for patients with Parkinson's disease. However, cell survival in brain tissue grafts is poor, with survival rates of only 5-15%. We have utilized a combination of the caspase inhibitor bocaspartyl (OMe)-fluoromethylketone (BOC-ASP-CH2F) and glial cell line-derived neurotrophic factor (GDNF) to enhance survival of grafted dopamine neurons. The VM tissue was dissected from embryonic day 13-15 rat fetuses, incubated in different doses of BOC-ASP-CH2F and GDNF, and transplanted to the anterior chamber of the eye of adult rats. Growth of the tissue was assessed through the translucent cornea. Doses of 50 and 100 micromolar of the general caspase inhibitor appeared to have detrimental effects on mesencephalic tissue, while 20 micromolar had beneficial effects on overall transplant growth. A combination of the caspase inhibitor and GDNF appeared to have more prominent effects on cell survival as well as dopaminergic fiber density than either agent by itself. The transplants doubled in size when they were treated with a combination of BOC-ASP-CH2F and GDNF, and cell death markers were significantly reduced at both 48 h and 4-6 days postgrafting. This is, to our knowledge, the first combined approach using apoptotic blockers with trophic factors, and demonstrates a viable strategy for protection of developing neurons, since several different aspects of graft function may be addressed simultaneously.

Animals↗

PubMed2000

Activation of atypical protein kinase C zeta by caspase processing and degradation by the ubiquitin-proteasome system.

Atypical protein kinase C zeta (PKCzeta) is known to transduce signals that influence cell proliferation and survival. Here we show that recombinant human caspases can process PKCzeta at three sites in the hinge region between the regulatory and catalytic domains. Caspase-3, -6, -7, and -8 chiefly cleaved human PKCzeta at EETD downward arrowG, and caspase-3 and -7 also cleaved PKCzeta at DGMD downward arrowG and DSED downward arrowL, respectively. Processing of PKCzeta expressed in transfected cells occurred chiefly at EETD downward arrowG and DGMD downward arrowG and produced carboxyl-terminal polypeptides that contained the catalytic domain. Epitope-tagged PKCzeta that lacked the regulatory domain was catalytically active following expression in HeLa cells. Induction of apoptosis in HeLa cells by tumor necrosis factor alpha plus cycloheximide evoked the conversion of full-length epitope-tagged PKCzeta to two catalytic domain polypeptides and increased PKCzeta activity. A caspase inhibitor, zVAD-fmk, prevented epitope-tagged PKCzeta processing and activation following the induction of apoptosis. Induction of apoptosis in rat parotid C5 cells produced catalytic domain polypeptides of endogenous PKCzeta and increased PKCzeta activity. Caspase inhibitors prevented the increase in PKCzeta activity and production of the catalytic domain polypeptides. Treatment with lactacystin, a selective inhibitor of the proteasome, caused polyubiquitin-PKCzeta conjugates to accumulate in cells transfected with the catalytic domain or full-length PKCzeta, or with a PKCzeta mutant that was resistant to caspase processing. We conclude that caspases process PKCzeta to carboxyl-terminal fragments that are catalytically active and that are degraded by the ubiquitin-proteasome pathway.

Animals↗

PubMed2000

Lipoproteins regulate expression of the steroidogenic acute regulatory protein (StAR) in mouse adrenocortical cells.

The steroidogenic acute regulatory protein (StAR) is required for the movement of cholesterol from the outer to the inner mitochondrial membrane, the site of cholesterol side chain cleavage. Here we describe a novel form of regulation of StAR gene expression in steroidogenic cells. Treatment of Y-1 BS1 adrenocortical cells with either low density lipoprotein (LDL) or high density lipoprotein (HDL) increases expression of endogenous StAR mRNA and protein in a dose-dependent manner. Induction of StAR mRNA by lipoprotein requires basal cAMP-dependent protein kinase, since the inhibitor, R(p)-8-Br-cAMP, inhibited induction of StAR protein by LDL. Likewise, basal StAR expression or LDL induction of StAR protein was not detectable in Y-1 kin-8 cells which are deficient in cAMP-dependent protein kinase. Aminoglutethimide and ketoconazole were used to determine if side chain cleavage of lipoprotein-derived cholesterol is required for induction of StAR mRNA. Treatment with either drug alone induced StAR mRNA expression 1.5-3-fold, while induction of StAR in cells treated with either drug plus LDL, was equal to, or greater than, induction seen with either agent alone, suggesting that lipoprotein does not regulate StAR via generation of an oxysterol intermediate. Both LDL and HDL increased expression of a mouse -966 StAR promoter-reporter construct 1.5-2.5-fold, indicating that regulation occurs at the level of transcription. In contrast, neither lipoprotein was able to induce transcription from a -966 StAR promoter in which the steroidogenic factor-1 site at -135 was abolished, indicating that regulation of StAR transcription by lipoproteins requires steroidogenic factor-1. The regulation of StAR gene expression by lipoproteins may represent a positive feedback circuit which links cholesterol availability with steroidogenic output.

Adrenal Cortex↗

PubMed2000

Activation of PKC is sufficient to induce an apoptotic program in salivary gland acinar cells.

Accumulating evidence suggests that specific isoforms of PKC may function to promote apoptosis. We show here that activation of the conventional and novel isoforms of PKC with 12-O-tetradecanoyl phorbol-13- ester (TPA) induces apoptosis in salivary acinar cells as indicated by DNA fragmentation and activation of caspase-3. TPA-induced DNA fragmentation, caspase-3 activation, and morphologic indicators of apoptosis, can be enhanced by pretreatment of cells with the calpain inhibitor, calpeptin, prior to the addition of TPA. Analysis of PKC isoform expression by immunoblot shows that TPA-induced downregulation of PKC alpha and PKC delta is delayed in cells pre-treated with calpeptin, and that this correlates with an increase of these isoforms in the membrane fraction of cells. TPA-induced apoptosis is accompanied by biphasic activation of the c-jun-N-terminal kinase (JNK) pathway and inactivation of the extracellular regulated kinase (ERK) pathway. Expression of constitutively activated PKC alpha or PKC delta, but not kinase negative mutants of these isoforms, or constitutively activated PKC epsilon, induces apoptosis in salivary acinar cells, suggesting a role for these isoforms in TPA-induced apoptosis. These studies demonstrate that activation of PKC is sufficient for initiation of an apoptotic program in salivary acinar cells. Cell Death and Differentiation (2000) 7, 1200 - 1209.

Animals↗

PubMed2000

Protein kinase C isozymes and the regulation of diverse cell responses.

Individual protein kinase C (PKC) isozymes have been implicated in many cellular responses important in lung health and disease, including permeability, contraction, migration, hypertrophy, proliferation, apoptosis, and secretion. New ideas on mechanisms that regulate PKC activity, including the identification of a novel PKC kinase, 3-phosphoinositide-dependent kinase-1 (PDK-1), that regulates phosphorylation of PKC, have been advanced. The importance of targeted translocation of PKC and isozyme-specific binding proteins (like receptors for activated C-kinase and caveolins) is well established. Phosphorylation state and localization are now thought to be key determinants of isozyme activity and specificity. New concepts on the role of individual PKC isozymes in proliferation and apoptosis are emerging. Opposing roles for selected isozymes in the same cell system have been defined. Coupling to the Wnt signaling pathway has been described. Phenotypes for PKC knockout mice have recently been reported. More specific approaches for studying PKC isozymes and their role in cell responses have been developed. Strengths and weaknesses of different experimental strategies are reviewed. Future directions for investigation are identified.

Animals↗

PubMed2000

Chronic hypoxia induces exaggerated growth responses in pulmonary artery adventitial fibroblasts: potential contribution of specific protein kinase c isozymes.

Enhanced proliferation of adventitial fibroblasts is a major contributor to the structural remodeling of the pulmonary artery (PA) that occurs during hypoxia-induced pulmonary hypertension. The mechanisms responsible for the exuberant growth of fibroblasts are unknown; however, protein kinase C (PKC) isozymes have previously been shown to be important in the enhanced growth properties of immature PA fibroblasts. We tested the hypotheses that PA adventitial fibroblasts from neonatal calves exposed chronically to hypoxia after birth would express augmented growth responses compared with fibroblasts from the control adventitia and that these properties would be associated with selective changes in expression of PKC isozymes. We studied the effects of serum, purified mitogens, and hypoxia on the growth of aggregate populations of fibroblasts isolated from the PA of neonatal control calves (Neo-C) and calves chronically exposed to hypoxia for 2 wk beginning on Day 1 of life (Neo-Hyp). Neo-Hyp fibroblasts demonstrated higher proliferative capabilities than did Neo-C cells in response to all the stimuli tested. Importantly, hypoxia was found to act synergistically with peptide mitogens (platelet-derived growth factor, basic fibroblast growth factor, insulin-like growth factor-I) to stimulate growth in Neo-Hyp but not in Neo-C cells. Using PKC-isozyme nonselective and selective inhibitors and immunoblot analysis, we found differences in utilization of PKC isozymes in Neo-Hyp and Neo-C fibroblasts and have identified PKC-betaI and -zeta as key contributors to the augmented growth of Neo-Hyp fibroblasts. Although the activity of PKC-betaI and -zeta isozymes was increased by hypoxia in serum-deprived Neo-C and Neo-Hyp fibroblasts, under normoxia, quiescent Neo-Hyp fibroblasts had higher PKC-zeta-specific activity than did Neo-C cells. These results suggest that neonatal PA adventitial fibroblasts acquire new growth properties in the setting of hypoxia- induced pulmonary hypertension and that the augmented proliferative characteristics of the Neo-Hyp fibroblasts might be associated with changes in specifc PKC isozyme expression and activation patterns.

Animals↗

PubMed1999

Protein kinase C delta is essential for etoposide-induced apoptosis in salivary gland acinar cells.

We have previously shown that parotid C5 salivary acinar cells undergo apoptosis in response to etoposide treatment as indicated by alterations in cell morphology, caspase-3 activation, DNA fragmentation, sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Here we report that apoptosis results in the caspase-dependent cleavage of protein kinase C-delta (PKCdelta) to a 40-kDa fragment, the appearance of which correlates with a 9-fold increase in PKCdelta activity. To understand the function of activated PKCdelta in apoptosis, we have used the PKCdelta-specific inhibitor, rottlerin. Pretreatment of parotid C5 cells with rottlerin prior to the addition of etoposide blocks the appearance of the apoptotic morphology, the sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Inhibition of PKCdelta also partially inhibits caspase-3 activation and DNA fragmentation. Immunoblot analysis shows that the PKCdelta cleavage product does not accumulate in parotid C5 cells treated with rottlerin and etoposide together, suggesting that the catalytic activity of PKCdelta may be required for cleavage. PKCalpha and PKCbeta1 activities also increase during etoposide-induced apoptosis. Inhibition of these two isoforms with Gö6976 slightly suppresses the apoptotic morphology, caspase-3 activation, and DNA fragmentation, but has no effect on the sustained activation of c-Jun N-terminal kinase or inactivation of extracellular regulated kinase 1 and 2. These data demonstrate that activation of PKCdelta is an integral and essential part of the apoptotic program in parotid C5 cells and that specific activated isoforms of PKC may have distinct functions in cell death.

Acetophenones↗

PubMed1999

Constitutive activation of the prolactin receptor results in the induction of growth factor-independent proliferation and constitutive activation of signaling molecules.

The ability to induce the oncogenic activation of the human prolactin receptor (PRLR) was examined by deleting 178 amino acids of the extracellular ligand-binding domain. Expression of this deletion mutant in the interleukin-3 (IL-3)-dependent murine myeloid cell line 32Dcl3 resulted in the induction of growth factor-independent proliferation. Parental 32Dcl3 cells proliferated only in the presence of exogenous murine IL-3 (mIL-3), while 32Dcl3 cells transfected with the long form of the human PRLR were able to proliferate in response to mIL-3, ovine prolactin, or human PRL. Cells expressing the Delta178 deletion mutant contained numerous phosphotyrosine-containing proteins in the absence of stimulation with either mIL-3 or ovine prolactin. Growth factor stimulation increased the number of proteins phosphorylated and the intensity of phosphorylation. These proteins included constitutively phosphorylated Janus kinase 2, signal transducer and activator of transcription 5, and SHC. Activated extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) were observed in unstimulated 32Dcl3 cells expressing the Delta178 mutant. Likewise, transfection of Nb2 cells with the Delta178 deletion mutant induced growth factor-independent proliferation and constitutive activation of Janus kinase 2, ERK1, and ERK2. In addition to the induction of a growth factor-independent state, the expression of the Delta178 deletion mutant also suppressed the apoptosis that occurs when 32Dcl3 cells are cultured in the absence of growth factors such as IL-3. These data suggest that the constitutive activation of the PRLR can be achieved by deletion of the ligand binding domain and that this mutation leads to the oncogenic activation of the receptor as determined by the ability of the receptor to induce growth factor-independent proliferation of factor-dependent hematopoietic cells.

Caseins↗

PubMed1999

Etoposide-induced activation of c-jun N-terminal kinase (JNK) correlates with drug-induced apoptosis in salivary gland acinar cells.

We have examined the ability of etoposide to induce apoptosis in two recently established rat salivary acinar cell lines. Etoposide induced apoptosis in the parotid C5 cell line as evidenced by the appearance of cytoplasmic blebbing and nuclear condensation, DNA fragmentation and cleavage of PARP. Etoposide also induced activation of c-jun N-terminal kinase (JNK) in parotid C5 cells by 4 h after treatment, with maximal activation at 8 - 10 h. Coincident with activation of JNK, the amount of activated ERK1 and ERK2 decreased in etoposide-treated parotid C5 cells. In contrast to the parotid C5 cells, the vast majority of submandibular C6 cells appeared to be resistant to etoposide-induced apoptosis. Likewise, activation of JNKs was not observed in etoposide-treated submandibular C6 cells, and the amount of activated ERK1 and ERK2 decreased only slightly. Etoposide treatment of either cell line had no effect upon the activation of p38. Treatment of the parotid C5 cells with Z-VAD-FMK, a caspase inhibitor, inhibited etoposide-induced activation of JNK and DNA fragmentation. These data suggest that etoposide may induce apoptosis in parotid C5 cells by activating JNKs and suppressing the activation of ERKs, thus creating an imbalance in these two signaling pathways.

Amino Acid Chloromethyl Ketones↗

PubMed1999

Vimentin-dependent utilization of LDL-cholesterol in human adrenal tumor cells is not associated with the level of expression of apoE, sterol carrier protein-2, or caveolin.

SW-13 adrenal tumor cells that lack detectable intermediate filaments (IF-free) exhibit an impaired capacity to esterify lipoprotein-derived cholesterol compared with cells that contain vimentin filaments. IF-free cells were found to synthesize and secrete significant amounts of apoE, while apoE secretion was nearly undetectable in cell lines that spontaneously express vimentin. However, stable transfectants that express a mouse vimentin cDNA exhibited elevated levels of cholesterol esterification and apoE secretion compared with untransfected IF-free cells, indicating that apoE secretion is not directly related to the capacity of these cells to esterify cholesterol. Some of the cell lines that differed in the level of apoE synthesis and secretion had similar levels of apoE mRNA, suggesting that the differences in expression involve a post-transcriptional mechanism. Treatment of these cells with forskolin and IBMX, 8br-cAMP, or TPA had no effect on apoE secretion. The level of sterol carrier protein-2 (SCP(2)) synthesis and the distribution of SCP(2) between membrane and soluble cellular fractions was not observably different in cells that contained or lacked vimentin. SW-13 cell lines contained little or no detectable caveolin-1 or caveolin-2. These studies demonstrate that the difference in the capacity of these adrenal tumor cells that contain or lack vimentin filaments to esterify low density lipoprotein-cholesterol is not obviously associated with the level of expression or distribution of apoE, SCP(2), or caveolins.

1-Methyl-3-isobutylxanthine↗

PubMed1998

Selective uptake of low density lipoprotein-cholesteryl ester is enhanced by inducible apolipoprotein E expression in cultured mouse adrenocortical cells.

Apolipoprotein (apo) E is expressed at high levels by steroidogenic cells of the adrenal gland, ovary, and testis. The cell surface location of apoE in adrenocortical cells suggests that apoE may facilitate the uptake of lipoprotein cholesterol by either the endocytic or the selective uptake pathways, or both. To examine these possibilities, the human apoE gene was expressed in murine Y1 adrenocortical cells under control of an inducible tetracycline-regulated promoter. The results show that induction of apoE yielded a 2-2.5-fold increase in the uptake of low density lipoprotein-cholesteryl ester (LDL-CE) but had little effect on high density lipoprotein-CE uptake. Analysis of lipoprotein uptake pathways showed that apoE increased LDL-CE uptake by both endocytic and selective uptake pathways. In terms of cholesterol delivery to the adrenal cell, the apoE-mediated enhancement of LDL-CE selective uptake was quantitatively more important. Furthermore, the predominant effect of apoE expression was on the low affinity component of LDL-CE selective uptake. LDL particles incubated with apoE-expressing cells contained 0.92 +/- 0.11 apoE molecules/apoB after gel filtration chromatography, indicating stable complex formation between apoE and LDL. ApoE expression by Y1 cells was necessary for enhanced LDL-CE selective uptake. This result may indicate an interaction between apoE-containing LDL and cell surface apoE. These data suggest that apoE produced locally by steroidogenic cells facilitates cholesterol acquisition by the LDL selective uptake pathway.

Adrenal Cortex↗

PubMed1998

Inducible expression of protein kinase Calpha suppresses steroidogenesis in Y-1 adrenocortical cells.

We have previously shown that protein kinase C (PKC) suppresses steroidogenesis in Y-1 adrenocortical cells. To ask directly if the PKCalpha isoform mediates this suppression, we have developed Y-1 cell lines in which PKCalpha is expressed from a tetracycline-regulated promoter. Induction of PKCalpha expression in these cell lines results in decreased P450 cholesterol side-chain cleavage enzyme (P450-SCC) activity as judged by the conversion of hydroxycholesterol to pregnenolone. Transcription of a P450-SCC promoter-luciferase construct is also reduced when PKCalpha expression is increased. However, expression of PKCalpha has no effect on 8-bromo-cAMP induction of steroidogenesis, indicating that these pathways function independently to regulate steroidogenesis. To determine the relationship between endogenous PKC activity and steroidogenesis, we examined 12 Y-1 subclones that were isolated by limited dilution cloning. In each of these subclones, steroid production correlates inversely with total PKC activity and with the expression of PKCalpha but not PKCepsilon or PKCzeta. These studies define for the first time the role of a specific PKC isoform (PKCalpha) in regulating steroidogenesis and P450-SCC activity in adrenocortical cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

Apolipoprotein E expression in Y1 adrenal cells is associated with increased intracellular cholesterol content and reduced free cholesterol efflux.

The expression of apoE mRNA in the adrenal gland is inversely correlated to steroidogenesis and directly correlated to the level of cholesteryl ester stores. To further investigate the relationship between apoE and cellular cholesterol homeostasis, several parameters of cholesterol metabolism in the murine Y1 adrenal cell line engineered to constitutively express human apoE (Y1-E cells) have been studied. It is reported here that Y1-E cells have increased cellular cholesterol content and markedly reduced efflux of free cholesterol as compared to control Y1 cells that do not express apoE. Y1-E cells have increases in both free and esterified cholesterol. However, Y1 and Y1-E cells incorporate [14C]oleate into cholesteryl ester at similar rates and have similar levels of maximal ACAT activity in isolated microsomes. Turnover of cholesteryl ester stores prelabeled with [14C]oleate occurred at similar rates in Y1-E and control Y1 cells, suggesting that increased cholesteryl ester stores in Y1-E cells do not result from reduced cholesteryl ester hydrolysis. Y1-E cells showed reduced cholesterol efflux as compared to control Y1 cells with either native high-density lipoprotein or cholesterol-free reconstituted particles as extracellular acceptors. Cholesterol efflux was not altered by inhibition of ACAT, suggesting that cholesterol esterification in Y1-E cells is not inhibiting efflux. These results suggest that reduced cholesterol efflux is responsible, at least in part, for the cholesterol accumulation in Y1-E cells. In comparison to the rat adrenal gland in vivo, Y1-E cells resemble adrenocortical cells under conditions where steroidogenesis is suppressed and apoE expression and cholesteryl ester storage are increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗

PubMed1993

Protein kinase C is a tonic negative regulator of steroidogenesis and steroid hydroxylase gene expression in Y1 adrenal cells and functions independently of protein kinase A.

The role of protein kinase C (PKC) in the regulation of basal steroidogenesis and steroid hydroxylase gene expression in Y1 adrenocortical cells was investigated. Treatment of Y1 cells with either staurosporine or calphostin C, inhibitors of PKC, increases steroid hormone production up to 7-fold. Induction of P450-cholesterol side chain cleavage enzyme (SCC) mRNA expression parallels induction of steroidogenesis by the PKC inhibitors. Staurosporine increases expression of a transiently transfected SCC promoter--human growth hormone construct in Y1 cells, indicating that PKC regulates expression of SCC mRNA at the level of transcription. Treatment with staurosporine increases expression of mRNA for two additional steroid synthetic enzymes, P450-11 beta-hydroxylase and 3 beta-hydroxysteroid dehydrogenase. These data indicate that PKC acts as a tonic negative regulator of basal steroidogenesis in Y1 cells by suppressing expression of mRNA encoding the steroid synthetic enzymes. Protein kinase A (PKA) and PKC have reciprocal effects on steroidogenesis and expression of the steroid synthetic enzymes in Y1 cells. However, the results of this study demonstrate that these signaling pathways are not interdependent. Steroid production by Y1 cells treated with (Bu)2cAMP and calphostin C together is equal to the sum of steroid production after treatment with either agent alone. Pretreatment of Y1 cells with Rp-8-Bromo-cAMP, a specific inhibitor of PKA, prevents induction of steroidogenesis by (Bu)2cAMP, but not by staurosporine, indicating that PKC is not dependent on PKA activity. In addition, induction of SCC mRNA expression by staurosporine, in Y1 cells which are defective in activation of PKA (Y1 kin-8), is equivalent to induction in Y1 cells. These data indicate that PKA and PKC regulate basal steroidogenesis through independent effects on expression of the steroid synthetic enzymes.

3-Hydroxysteroid Dehydrogenases↗

PubMed1992

Elevated levels of protein kinase C in Y1 cells which express apolipoprotein E decrease basal steroidogenesis by inhibiting expression of P450-cholesterol side chain cleavage mRNA.

We have previously reported that steroidogenesis is dramatically reduced in mouse Y1 adrenocortical cells which express the human apolipoprotein E gene (Y1-E cells). This suppression results in part from inhibition of cAMP-mediated events. In this report we have examined the expression of protein kinase C (PKC) in the Y1-E cell lines. Total cellular PKC activity in vitro is increased 3-5-fold in the Y1-E cell lines. PKC activity in the particulate and cytosolic fractions is increased to the same relative extent. Increased PKC activity reflects increased levels of PKC mRNA, as determined by Northern blot analysis, and PKC protein, as determined by immunoblot analysis. Increased expression of PKC in the Y1-E cell lines is accompanied by a 2-3-fold increase in diacylglycerol, an in vivo activator of PKC. To determine the contribution of elevated PKC expression to the Y1-E cell phenotype, we utilized the PKC inhibitors, staurosporine and calphostin C. Upon treatment with staurosporine or calphostin C, expression of P450-cholesterol side chain cleavage mRNA is increased severalfold to a level equal to, or greater than, basal expression in the Y1-neo control cell line. Treatment with calphostin C also results in recovery of steroidogenesis in the Y1-E cells to a level comparable to the basal level observed in the Y1-neo control cell line. These results indicate that increased expression of PKC in the Y1-E cell lines decreases basal steroidogenesis by suppressing P450-cholesterol side chain cleavage mRNA expression. Inhibition of PKC, however, does not reverse the block in cAMP-stimulated steroidogenesis in Y1-E cells, suggesting that the pleiotropic effects of apoE expression are not mediated entirely through altered PKC expression.

Adrenal Cortex↗

PubMed1991

Suppression of cAMP-mediated signal transduction in mouse adrenocortical cells which express apolipoprotein E.

We have reported previously that expression of the human apolipoprotein E (apoE) gene in mouse Y1 adrenocortical cells suppresses basal and adrenocorticotropin (ACTH)-stimulated steroidogenesis. To understand the mechanism of this suppression, we have examined the integrity of cAMP regulated events required for adrenal steroidogenesis. Both acute and chronic responses to ACTH or cAMP are suppressed in Y1 cells which express apoE (Y1-E cells) as compared with parental Y1 cells. Acute morphologic changes in response to cAMP and acute induction of steroidogenesis by cAMP are suppressed in the Y1-E cell lines. Constitutive expression of P450-cholesterol side chain cleavage enzyme mRNA, the rate-limiting enzyme in steroid hormone synthesis, is reduced up to 11-fold in the Y1-E cell lines. The level of mRNA encoding P450-cholesterol side chain cleavage correlates directly with the reduction in basal steroid production observed in the individual Y1-E cell lines. Expression of P450-11 beta-hydroxylase mRNA, although readily detectable in Y1 parent cells, is absent or reduced in the Y1-E cell lines. Inhibition of cAMP-regulated gene expression is not restricted to genes required for steroid synthesis, since cAMP induction of ornithine decarboxylase mRNA is also inhibited in the Y1-E cell lines. These data indicate that suppression of steroidogenesis in Y1-E cells is due, at least in part, to inhibition of cAMP-regulated gene expression. These effects are not due to a defective cAMP-dependent protein kinase, since kinase activity in vitro and activation in vivo are unaltered in the Y1-E cell lines. These results suggest that expression of apoE in Y1 cells blocks cAMP-mediated signal transduction at a point distal to activation of cAMP-dependent protein kinase.

Adrenal Cortex↗