Effects of interleukin-1 beta on secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by cultured rat anterior pituitary cells

Interleukin-1 beta (IL-1 beta) at doses of 0.15 and 1.5 nM significantly inhibited FSH secretion and stimulated LH secretion by cultured rat pituitary cells after 24-72 hr incubation whereas 15 pM of IL-1 beta was not effective. Treatment with IL-1 beta for 12-48 hr did not affect intracellular content of FSH. However, treatment with 0.15 and 1.5 nM of IL-1 beta for 72 hr significantly suppressed intracellular content of FSH whereas various doses of IL-1 beta incubated with the cells for 12-72 hr showed no effect on the intracellular content of LH. Pretreatment with IL-1 beta for 48 hr inhibited both GnRH-mediated LH and FSH secretions by the pituitary. The secretion of FSH and LH mediated by an activator of protein kinase C, phorbol 12-myristate 13-acetate, was also significantly suppressed by pretreatment with IL-1 beta for 48 hr. These results suggest that (a) IL-1 beta has opposite effects on the secretion of LH and FSH and (b) pretreatment with IL-1 beta suppresses GnRH-mediated stimulation of LH and FSH by the pituitary and this suppressive effect of IL-1 beta may involve the suppression of a protein kinase C-dependent mechanism.     

Regulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase activity and regulatory subunit RII beta content by basic fibroblast growth factor (bFGF) during granulosa cell differentiation: possible implication of protein kinase C in bFGF action.

We have previously shown that basic fibroblast growth factor (bFGF) inhibits the FSH-induced differentiation of cultured rat granulosa cells, as manifested by prominent reduction of the LH receptor expression. We now investigate the possible sites and mechanism of action of bFGF. Whereas bFGF decreased the cAMP formation induced by FSH, it enhanced the cAMP production caused by cholera toxin and forskolin, suggesting that bFGF exerted its inhibitory action on cell differentiation at a step to cAMP production. Photoaffinity labeling with 8-azido-[32P]cAMP revealed that bFGF markedly reduced the FSH-induced increase in the level of regulatory subunit RII beta of the cAMP-dependent protein kinase (PKA) type II. In contrast to its striking effect on RII beta expression (70-80% inhibition), bFGF decreased PKA enzymatic activity by only 30%. On the other hand, transforming growth factor-beta (TGF beta) slightly amplified the stimulatory action of FSH and antagonized the bFGF inhibitory effect on both LH receptor expression and RII beta synthesis. We report that the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA), which impaired granulosa cell differentiation, also abolished the RII beta synthesis induced by FSH. The activation of PKC by bFGF in granulosa cells was supported by the following findings: (i) bFGF markedly enhanced the production of diacylglycerol (2.3-fold stimulation at 5 min), the intracellular activator of PKC; (ii) bFGF promoted tight association of PKC to cellular membranes, a process that is believed to correlate with the enzyme activation; (iii) bFGF induced the phosphorylation of an endogenous M(r) 78,000/pI 4.7 protein that appears as a specific PKC substrate; (iv) bFGF mimicked the TPA-induced transmodulation of the epidermal growth factor (EGF) receptor, reducing by 36% the 125I-EGF binding on granulosa cells. We conclude that bFGF may exert its repressive action on RII beta synthesis, PKA activity, and granulosa cell differentiation by primarily targeting PKC activation.     

Selective activation of the gamma-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites

The gamma-subspecies of protein kinase C (PKC) apparently is expressed only in central nervous tissues, and at a high level in the cerebellum and hippocampus. gamma-PKC from bovine cerebellum, but not the alpha- or beta I/beta II-subspecies, is activated by micromolar concentrations of arachidonic acid (AA), in the absence of both phospholipid and diacylglycerol. A significant component of this activation is also calcium independent. Other unsaturated fatty acids are much less active in this respect. Among the AA metabolites tested, lipoxin A (5(S),6(R),15(S)-11-cis-isomer) was a potent, selective activator of the gamma-subspecies, and also, to a lesser extent, 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid could support activation. These results raise the possibility that AA and some of its lipoxygenase metabolites may function as messenger molecules in neurones to activate the gamma-subspecies of PKC.     

Suppression of interleukin-1 beta and LDL scavenger receptor expression in macrophages by a selective protein kinase C inhibitor.

A human monocytic cell line, THP-1, stimulated with 40 nM phorbol myristate acetate (PMA), differentiated to macrophage-like cells, and exhibited increased expression and release of interleukin-1 beta and expression of acetylated low density lipoprotein (ac-LDL) receptors. A selective inhibitor, MDL 29,152 (4-propyl-5-(4-quinolinyl)-2(3H)-oxazolone) was used to show that this induction required activation of protein kinase C. MDL 29,152 acts in the catalytic domain of protein kinase C and is at least 200-fold selective for protein kinase C over cAMP-dependent protein kinase in THP-1 cells. MDL 29,152 (50 microM) reduced levels of interleukin-1 beta mRNA in PMA-stimulated cells by 76% and eliminated detectable interleukin-1 beta in the media. Flow cytometric analysis showed that 48 h after THP-1 activation, approximately 50% of the cells expressed ac-LDL receptors, while in the presence of 100 microM MDL 29,152, less than 5% of the cells expressed receptors. The relationship between THP-1 differentiation and protein kinase C activation was determined by following the expression of the cell surface antigen MO-1. Expression of MO-1 antigen increases as monocytes differentiate to macrophages. After 48 h of phorbol activation, 90% of the THP-1 population was MO-1-positive; less than 16% of the population was MO-1-positive when 100 microM MDL 29,152 was present. By dual analysis, it was found that within the differentiated, MO-1-positive population, only approximately 50% of the cells also expressed ac-LDL receptors. Based on these findings, we conclude that protein kinase C promotes processes important in THP-1 activation and differentiation to macrophage-like cells including interleukin-1 beta expression and secretion, ac-LDL receptor and MO-1 expression.     

Protein kinase C isozyme expression in phorbol ester-sensitive and -resistant EL4 thymoma cells

To investigate whether differential protein kinase C isozyme expression in phorbol ester-sensitive and -resistant EL4 thymoma cells could account for the difference in phorbol ester responsiveness, we purified and characterized isozymes from the two cell lines. In both cell types, two peaks of protein kinase C activity were resolved on hydroxylapatite following DEAE-cellulose and phenyl-Superose chromatography. Western blot analysis showed that the first peak corresponded to protein kinase C-beta and the second to protein kinase C-alpha. Two-dimensional phosphotryptic mapping of the purified alpha and beta isozymes did not reveal any reproducible differences between sensitive and resistant EL4 cells. Nor were any differences between the cell types observed in the cytosolic versus membrane localization of alpha and beta protein kinase C. Northern blot analysis showed the expression of mRNA for protein kinase C-alpha, -beta, -delta and -epsilon in both cell lines, and the absence of mRNA for gamma or zeta. Although no major differences in expression of alpha, beta, or delta mRNA between sensitive and resistant EL4 cells were detectable, expression of protein kinase C-epsilon mRNA in resistant cells was only 20-25% of that in sensitive. Western blot analysis with anti-protein kinase C-epsilon antibodies showed the presence of the epsilon-isozyme in sensitive cells and the absence of detectable amounts in resistant cells. Although protein kinase C-epsilon constitutes only a small portion of the total protein kinase C in sensitive cells, the possibility is raised that decreased protein kinase C-epsilon expression may contribute to the failure of resistant EL4 cells to respond to phorbol esters.     

Hormonal regulation of tissue-type plasminogen activator messenger ribonucleic acid levels in rat granulosa cells: mechanisms of induction by follicle-stimulating hormone and gonadotropin releasing hormone

FSH and GnRH both stimulate rat granulosa cells to produce tissue-type plasminogen activator (tPA). We have studied the molecular mechanisms involved in the action of these hormones by measuring tPA mRNA levels in primary cultures of rat granulosa cells. When granulosa cells were cultured in the presence of FSH or GnRH the level of tPA mRNA was increased 20- and 12-fold, respectively. The induction of tPA mRNA by FSH and GnRH was additive and the kinetics of induction differed. The effect of FSH could be mimicked by bromo-cAMP or forskolin, and was drastically enhanced by cotreatment with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. These findings are consistent with the notion that FSH mediates its effect through the protein kinase A pathway. GnRH is believed to augment phospholipid turnover in granulosa cells, leading to the activation of the protein kinase C pathway. Like GnRH, the protein kinase C activator phorbol myristate acetate also induced tPA mRNA in granulosa cells. In the presence of the protein synthesis inhibitor, cycloheximide, FSH-stimulated tPA message levels were enhanced by 30-fold, revealing superinduction of tPA mRNA levels by this pathway. In contrast the induction of tPA mRNA by GnRH was inhibited by cycloheximide indicating that the synthesis of an intermediate protein is required for the GnRH effect. Our data suggest that FSH and GnRH increase the tPA mRNA levels by two distinct pathways in cultured granulosa cells, providing a model system for studying the hormonal regulation of tPA gene expression.     

Phorbol diester-induced alterations in the expression of protein kinase C isozymes and their mRNAs. Analysis in wild-type and phorbol diester-resistant HL-60 cell clones.

In an HL-60 cell subline (PR-17) which was greater than 100-fold resistant to the differentiating and cytostatic activities of phorbol 12-myristate 13-acetate (PMA), the protein kinase C phenotype was found to be nearly identical to that of wild-type HL-60 cells. A measurable decrease (30%) in the specific activities of crude preparations of PR-17 cell protein kinase C was observed when the enzyme was measured with histone as the phosphate acceptor substrate, but other aspects of the protein kinase C phenotype (intracellular concentrations and binding affinities of phorbol diester receptors, translocation of activated enzyme from cytosolic to particulate subcellular fractions, relative expression of the alpha and beta isozyme proteins) were equivalent in both PMA-resistant PR-17 cells and in wild-type HL-60 cells. Direct analysis of the behavior of the alpha and beta isozymes after the exposure of each cell type to 100 nM PMA for 12 h revealed that the activities and intracellular concentrations of both isozymes were downregulated to an equivalent extent in both wild-type and PMA-resistant cells. These results suggest that the cellular basis for the resistance to the effects of PMA was present "down-stream" from the activation and down-regulation of protein kinase C and was perhaps a nuclear component. Among the genes which were likely to be differentially regulated when each of the two cell lines were treated with PMA were those for the protein kinase C isozymes themselves. In wild-type HL-60 cells, the intracellular concentrations of type HL-60 cells, the intracellular concentrations of mRNA for each of the beta isozymes were increased (up to 5-fold) 48 h after the initiation of PMA treatment; further studies indicate that an activator of protein kinase C could influence the expression of HL-60 cell protein kinase C genes in an isozyme-specific manner. Comparable PMA-induced alterations in mRNA levels were not observed in PMA-resistant cells, even under conditions of significant activation and subsequent down-regulation of protein kinase C protein. Taken together, these data suggest that activation and down-regulation of the isozymes of protein kinase C may not represent absolute determinants of the PMA-induced differentiation of HL-60 cells, but that specific alterations in the levels of the mRNA for the beta isozymes of protein kinase C, or of other genes which may be regulated by the activated kinase isozymes, are important to the induction of leukemia cell differentiation by PMA.     

Functional and subcellular changes in the A-kinase-signaling pathway: relation to aromatase and Sgk expression during the transition of granulosa cells to luteal cells.

The responsiveness of granulosa cells to FSH (cAMP) changes as these cells switch from the proliferative stage in growing follicles to the terminally differentiated, nonproliferating stage after LH-induced luteinization. To analyze this transition, two well characterized culture systems were used. 1) Granulosa cells isolated from immature rats were cultured in serum-free medium, a system that permits analysis of dynamic, short-term responses to hormones/cAMP. 2) Granulosa cells from preovulatory (PO) follicles that have been exposed in vivo to surge concentrations of hCG (PO/ hCG) were cultured in medium containing 1% FBS, a system that permits analyses of cells that have undergo irreversible, long-term changes associated with luteinization. To analyze the biochemical basis for the switch in cAMP responsiveness, the localization of A-kinase pathway components was related to the expression of two cAMP target genes, aromatase (CYP19) and serum-and glucocorticoid-induced kinase (Sgk). Components of the A-kinase pathway were analyzed by Western blotting and indirect immunofluorescence using specific antibodies to the C subunit, RIIalpha/beta subunits, CREB (cAMP-regulatory element binding protein), phospho-CREB, CBP (CREB binding protein), and Sgk. Cellular levels of C subunit and CREB were similar in all cell types and hormone treatments. CREB and CBP were nuclear; RIIalpha/beta was restricted to a cytoplasmic basket-like structure. Addition of FSH to immature granulosa cells caused rapid nuclear import of C subunit within 1 h. Nuclear C subunit decreased by 6 h after FSH but could be rapidly reimported to the nucleus by the addition of forskolin at 6, 24, or 48 h. Nuclear C subunit was associated with the rapid but transient increases in phospho-CREB. FSH induced Sgk in a biphasic manner in which the protein was nuclear at 1 h and cytoplasmic at 48 h. Aromatase mRNA was only expressed at 24-48 h after FSH, a pattern that was not altered by phosphodiesterases or phosphatases. In the luteinized (PO/hCG) granulosa cells, immunoreactive C subunit was localized in a punctate pattern in the nucleus as well as to a cytoplasmic basket-like structure, a distribution pattern not altered by forskolin. Aromatase, Sgk, and phospho-CREB were expressed at elevated levels in a non-forskolin-responsive manner. Most notable, both phospho-CREB and Sgk were preferentially localized in a punctate pattern within the cytoplasm and not altered by forskolin. Collectively, these data indicate that when granulosa cells differentiate to luteal cells the subcellular localization (nuclear vs. cytoplasmic) of A-kinase pathway components changes markedly. Thus, either the mechanisms of nuclear import and export or the presence of distinct docking sites (and functions ?) dictate where A-kinase, phospho-CREB and Sgk are localized in granulosa cells compared with the terminally differentiated luteal cells.     

Activation of distinct protein kinase C isozymes by phorbol esters: correlation with induction of interleukin 1 beta gene expression

Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., & Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1 beta (IL-1 beta) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 X 10(-8) M TPA. Higher concentrations of TPA, which partially or totally depleted protein kinase C levels in the cells (10(-9)-2 X 10(-5) M), had an inhibitory effect on IL-1 beta mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1 beta mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 X 10(-8) M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the alpha, beta, and gamma isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC alpha was particulate, and PKC beta was cytosolic, while there was no detectable PKC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of follicle-stimulating hormone-receptor messenger RNA in hen granulosa cells relative to follicle selection

Both the viability of hen prehierarchal follicles and subsequent differentiation associated with the selection of a single follicle per day into the preovulatory hierarchy depend on circulating FSH and the expression of FSH receptor (FSH-R) in granulosa cells. The present study addresses mechanisms that mediate both basal expression plus selective up-regulation of FSH-R mRNA in granulosa cells from prehierarchal follicles. Results demonstrate that FSH-R mRNA is both expressed and functional in granulosa cells collected from growing prehierarchal follicles as small as those of 1-2 mm in diameter, as indicated by rapid induction of steroidogenic acute regulatory (StAR) protein expression by FSH in vitro. Real-time polymerase chain reaction determined that relative FSH-R expression within the granulosa layer from individual prehierarchal follicles of 6-8 mm in diameter was similar among the 8-13 follicles within this cohort, with the notable exception that the granulosa layer from a single follicle (presumably the selected follicle) showed elevated expression. Levels of FSH-R mRNA expression were enhanced by both recombinant human (rh) transforming growth factor (TGF) beta1 and, to a lesser extent, rh-activin A after 20 h of culture. This stimulatory effect was effectively blocked by mitogen-activated protein (MAP) kinase signaling induced by TGF alpha treatment. Finally, inhibition of MAP kinase signaling, using the selective inhibitor U0126, promoted FSH-R expression and further enhanced TGF beta1-induced FSH-R expression in vitro. Collectively, results suggest that premature granulosa cell differentiation normally is suppressed by tonic MAP kinase signaling. At the time of follicle selection, a release from inhibitory MAP kinase signaling is proposed to occur, which enables the full potentiation of FSH-R expression mediated by intrafollicular factors.     

Affinity purification of the C alpha and C beta isoforms of the catalytic subunit of cAMP-dependent protein kinase

A synthetic peptide of 18 amino acids corresponding to the inhibitory domain of the heat-stable protein kinase inhibitor was synthesized and shown to inhibit both the C alpha and C beta isoforms of the catalytic (C) subunit of cAMP-dependent protein kinase. Extracts from cells transfected with expression vectors coding for the C alpha or the C beta isoform of the C subunit required 200 nM protein kinase inhibitor peptide for half-maximal inhibition of kinase activity in extracts from these cells. An affinity column was constructed using this synthetic peptide, and the column was incubated with protein extracts from cells overexpressing C alpha or C beta. Elution of the affinity column with arginine allowed single step isolation of purified C alpha and C beta subunits. The C alpha and C beta proteins were enriched 200-400-fold from cellular extracts by this single step of affinity chromatography. No residual inhibitory peptide activity could be detected in the purified protein. The purified C subunit isoforms were used to demonstrate preferential antibody reactivity with the C alpha isoform by Western blot analysis. Furthermore, preliminary characterization showed both isoforms have similar apparent Km values for ATP (4 microM) and for Kemptide (5.6 microM). These results demonstrate that a combination of affinity chromatography employing peptides derived from the heat-stable protein kinase inhibitor protein and the use of cells overexpressing C subunit related proteins may be an effective means for purification and characterization of the C subunit isoforms. Furthermore, this method of purification may be applicable to other kinases which are known to be specifically inhibited by small peptides.     

Enhanced expression of the beta II subspecies of protein kinase C in differentiating erythroleukemia cells

Polyclonal antipeptide antibodies which recognize selected isozymes (alpha, beta I, beta II, and gamma) of the protein kinase C family were used to identify specific subspecies in undifferentiated Friend erythroleukemia cells and in cells triggered to differentiate with hexamethylene bisacetamide. The beta II isozyme of protein kinase C was the primary isozyme expressed and its abundance was significantly increased (P less than 0.05) in differentiated cells. Differences in immunostaining between control and experimental groups were objectively quantitated by determining percentage transmission of light through cells based on color threshold rather than gray intensity levels. Staining was localized to the cytoplasm predominantly in differentiated cells, whereas nuclei stained more intensely in undifferentiated cells. These results provide immunocytochemical evidence to support the hypothesis that changes in the expression of the beta II subspecies of protein kinase C are essential to the programmed maturation of differentiating Friend erythroleukemia cells.     

Permeabilization by streptolysin-o reveals a role for calcium-dependent protein kinase c isoforms alpha and beta in the response of cultured cardiomyocytes to hyposmotic challenge

Immunocytochemical techniques indicate that the uninhibited activity of protein kinase C alpha and protein kinase C beta are necessary for a normal regulatory volume decrease (RVD) response of cultured chick embryo cardiomyocytes subjected to a hyposmotic environment. Antibodies against protein kinase C isoforms alpha, beta, gamma and epsilon were introduced into the cultured myocytes using a developed streptolysin-O (SLO) permeabilization technique that allows the targeted cells to accumulate large biomolecules without perturbing their normal physiological state. The loaded cells were then tested for their ability to RVD when submitted to hypo-osmotic stimulus. Results show that exposing the cultured cells to SLO in the presence of antibodies against protein kinase C alpha and beta, prior to volume challenge, significantly slows the RVD rate. Additional experiments that combined anti-alpha and anti-beta antibodies in the same exposure media did not result in a significantly different rate than the anti-alpha or anti-beta rates alone. The evidence gained in this study is in agreement with previous work in the cultured chick embryo cardiomyocyte that report the involvement of a calcium dependent protein kinase C in the signal transduction pathway of the RVD.     

An inhibitory role for the protein kinase C pathway in ovarian steroidogenesis. Studies with cultured swine granulosa cells.

We have used primary cultures of swine granulosa cells to investigate the regulatory role of the protein kinase C pathway in the ovary. In this system, we observed the following. Swine granulosa cells bound [3H]phorbol 12,13-dibutyrate [( 3H]PDB) specifically with high affinity [apparent Ki for 12-O-tetradecanoylphorbol 13-acetate (TPA) = 3.1 (2.1-4.7) nM] and low capacity [0.68 (0.34-0.99) pmol/10(7) cells]. The cytosol of granulosa cells contained functionally active protein kinase C capable of phosphorylating distinct proteins in response to stimulation with active phorbol ester. TPA and PDB induced dose-dependent inhibition (greater than 85%) of follicle-stimulating-hormone (FSH)-stimulated progesterone production. Half-maximally inhibitory concentrations were 0.10 and 0.75 nM for TPA and PDB respectively, whereas phorbol analogues that do not activate protein kinase C were not inhibitory. TPA did not impede cyclic AMP generation in response to FSH, cholera toxin or forskolin acutely (within 48 h), but did inhibit the stimulatory effects of 8-bromo cyclic AMP, insulin and oestradiol on progesterone biosynthesis. In the presence of maximally effective concentrations of 25-hydroxy-, 20 alpha-hydroxy- or 22R-hydroxy-cholesterol as exogenous sterol substrates for cholesterol side-chain cleavage, treatment with TPA suppressed pregnenolone, progesterone and 20 alpha-hydroxypregn-4-en-3-one biosynthesis by more than 80%. The inhibitory effects of phorbol esters were not attributable to non-specific cytotoxicity, since prostaglandin F2 alpha production increased in the same cultures and aromatization of exogenously supplied testosterone to oestradiol was not suppressed. In intact granulosa cells, the effects of phorbol esters were mimicked by a synthetic non-diterpene diacylglycerol, 1-octanoyl-2-acetylglycerol, and the tumour promoter, mezerein, which specifically activates protein kinase C. We conclude that swine granulosa cells contain specific high-affinity receptors for phorbol esters that are functionally coupled to protein phosphorylation. Moreover, treatment with phorbol esters or non-phorbol activators of protein kinase C results in selective inhibition of cholesterol side-chain cleavage activity without impairing cyclic AMP generation or oestrogen biosynthesis.     

Mechanism of the stimulating effect of estradiol on protein kinase C in plasma membranes of target cells

Using inhibitors and activators of protein kinase C, it was demonstrated that in isolated plasma membranes of target cells estradiol-17 beta selectively stimulates protein phosphorylation by endogenous protein kinase C. In estradiol-dependent tissues, estradiol effectuates the translocation of protein kinase C from the cytosol to the membrane fraction within 10-12 minutes. Estradiol activates protein kinase C in cellular membranes of target tissues via a mechanism which is different from that of phorbol ester (TPA): 3H-estradiol, in contrast with 3H-TPA, it is not bound by protein kinase C and, in contrast with TPA, estradiol-17 beta does not activate purified protein kinase C in vitro. In this case, the specific stimulation of protein kinase C translocation to membranes and the estradiol-induced increase in the phosphorylation of plasma membrane proteins seem to be due to the estradiol-induced activation of the transmembrane system of polyphosphoinositide degradation, eventually resulting in the formation of diacylglycerol, a protein kinase C activator.     

Regulation of C－Fos mRNA Expression in Sertoli Cells by cAMP，Ca~（＋＋），and protein Kinase C－mediated Pathways

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