Membrane-associated Protein Kinase C Activity Research Articles

Growth-dependent Accumulation of Monoalkylglycerol in Madin-Darby Canine Kidney Cells: EVIDENCE FOR A ROLE IN THE REGULATION OF PROTEIN KINASE C

1-O-Alkyl-sn-glycerol (alkylglycerol) forms the backbone of complex ether-linked glycerolipids, including biologically active lipids such as platelet-activating factor. Synthetic alkylglycerol itself possesses several potent pharmacological activities and has been shown to inhibit protein kinase C (PKC) in vitro. In spite of these properties, free alkylglycerol has been regarded only as a potential product of the inflammatory degradation of complex ether lipids rather than a natural cell constituent. To explore the possibility that endogenous alkylglycerol functions as a physiological regulator in normal cells, we measured its content, along with related monoglycerides and diglycerides, by high performance liquid chromatography and gas-liquid chromatography in Madin-Darby canine kidney (MDCK) cells. The content of free alkylglycerol increased up to 20-fold during the growth of MDCK cell cultures to a confluent density. The increase was greatest during the log phase of growth, in which the content of alkylglycerol rose from 6.0 +/- 1.3 nmol/10(8) cells in preconfluent cultures to 23.6 +/- 3.4 nmol/10(8) cells in confluent cultures. Analysis of the molecular species of alkylglycerol showed that the higher content in quiescent MDCK cells was due primarily to an increase in 1-O-octadecyl-sn-glycerol. In contrast, the levels of monoacylglycerol and the PKC activator diacylglycerol were lower in confluent, quiescent cultures than in preconfluent, proliferating cultures. A similar pattern of changes in the monoglyceride and diglyceride content was observed in interleukin-3-dependent CFTL-12 mast cells when cell proliferation was blocked by growth factor withdrawal. Growth of MDCK cells to a confluent density resulted in a decrease in particulate PKC enzyme activity to a level that was only 6% of that in proliferating cells. To explore whether the accumulation of cellular alkylglycerol contributes to growth-dependent changes in PKC activity, we examined the effects of adding alkylglycerol to the activity and subcellular distribution of the enzyme in MDCK cells. Treatment of cells with 1-O-dodecyl-sn-glycerol resulted in a decrease in the activity of membrane-associated PKC activity and inhibited 12-O-tetradecanoylphorbol-13-acetate-stimulated translocation of PKC from the cytosol to the membrane fraction. Alkylglycerol was also shown to inhibit the activity of purified PKC in vitro when present at levels similar to that of the diacylglycerol activator. We propose that the accumulation of alkylglycerol during the growth of MDCK cells to a confluent density contributes to the decrease in PKC activity. The control of cellular alkylglycerol levels may be a novel mechanism for the regulation of cellular physiology.

Long-lasting increase in protein kinase C activity in the hippocampus of amygdala-kindled rat

Previous studies have demonstrated that membrane-associated protein kinase C (PKC) activities in the right and left hippocampus of rats kindled from the left hippocampus increased significantly at 4 weeks [9] and 4 months [22] after the last seizure compared with those in matched control rats. In this study, we investigated the effect of kindling from the left amygdala on PKC activities in the amygdala/pyriform cortex and hippocampus at long seizure-free intervals (4 and 16 weeks) from the last amygdala-kindled seizure. Membrane-associated PKC activity of the kindled group increased significantly only in the left hippocampus compared with the left side control (the left hippocampus of rats subjected to a sham operation) at 4 weeks (by 34%, P < 0.03) and 16 weeks (by 24%, P < 0.05) after the last seizure. There was no significant alteration in the membrane-associated PKC activity of the kindled group in the right hippocampus or amygdala/pyriform cortex in any seizure-free interval after the last amygdala seizure. Cytosolic PKC activity did not differ between the kindled and control groups in any brain region examined in any seizure-free interval. At 16 weeks after the last seizure, the PKC activity in the P 1 fraction of the kindled group increased significantly only in the left hippocampus (by 49%, P < 0.005), but not in the right hippocampus. Neither PKC activity in the P 2 fraction nor that in the cytosolic fraction was altered in the kindled group after this seizure-free interval. The prolonged increase in activity of the membrane-associated PKC and that in the P 1 fraction in the hippocampus induced by amygdala-kindling may contribute to long-lasting seizure susceptibility induced by kindling.

CD28 cross-linking augments TCR-mediated signals and costimulates superantigen responses.

The CD28 molecule expressed on the surface of T cells plays a pivotal role in transducing costimulatory signals necessary for cell activation. CD28 coligation enhances tyrosine phosphorylation and phosphoinositol 3-kinase association in responsive cells. CD28 cross-linking has also been reported to activate inositol phospholipid turnover and to cause release of intracellular calcium. Here we examine the effects of CD28 cross-linking on early activation of protein kinase C (PKC). We have reported recently that either PMA or CD28 cross-linking synergizes with signals delivered by superantigen and cytokines to induce the proliferation of APC-depleted T cells. Unlike PMA, CD28 cross-linking alone failed to induce an increase in membrane-associated PKC activity. However, PKC activation was seen in resting T cells when CD28 was cross-linked in the presence of superantigen plus APC-derived supernatant, which by themselves had no effect on PKC activity. Inhibition of PKC activity using calphostin C blocked the response of pure T cells to superantigen in the presence of either autologous APC, PMA, or CD28 cross-linking. This effect was specific; it was only seen when calphostin C was added within the first hour of stimulation. Assays of [Ca2+]i levels showed that CD28 cross-linking augmented and prolonged the rise in [Ca2+]i induced in T cells by superantigen and APC-derived cytokines. In the presence of superantigen, the proliferative response of T cells costimulated by CD28 cross-linking was cyclosporin A-sensitive, whereas in the presence of PMA, CD28 cross-linking conferred resistance to cyclosporin A. Both the phosphorylation of phospholipase C gamma 1 at tyrosine and the rise in [Ca2+]i induced by CD28 cross-linking in preactivated T cells were blocked by herbimycin A. Herbimycin A treatment also blocked the ability of CD28 cross-linking to induce a rise in [Ca2+]i in resting T cells. We conclude that CD28 costimulatory signals augment superantigen-induced TCR signals by converging onto common TCR effector pathways involving the activation of phospholipase C gamma 1 and PKC and by generating a cyclosporin A-sensitive pathway.

Effect of dexamethasone and prednisolone on insulin-induced activation of protein kinase C in rat adipocytes and soleus muscles

We examined the effect of glucocorticoids on [ 3H]2-deoxyglucose ([ 3H]2-DOG) uptake, [ 125I]insulin binding, tyrosine kinase activity, and protein kinase C (PKC) activity in rat adipocytes and soleus muscles. In adipocytes, insulin-stimulated [ 3H]2-DOG uptake was decreased by prior 60-minute treatment with dexamethasone (DEX) or prednisolone (PSL), whereas [ 125I]insulin binding, insulin (INS) receptor autophosphorylation, and tyrosine kinase activity, as measured using exogenous substrate of poly(Glu 80-Tyr 20), were not significantly changed. Cytosolic PKC activity decreased and membrane-associated PKC activity increased during a 60-minute treatment of adipocytes and soleus muscles with DEX or PSL, indicating that both DEX and PSL stimulate the translocation and activation of PKC. However, pretreatment of adipocytes and soleus muscles with glucocorticoids resulted in reduced INS-stimulated translocation of PKC from cytosol to membrane. INS-induced decreases in cytosolic PKC activity (50% ± 7% v 10% ± 8% and 20% ± 7%, P < .05 to .01, for nonpretreated [control], DEX pretreated, and PSL pretreated cells) and increases in membrane PKC (100% ± 10% v 50% ± 9% and 20% ± 9%, P < .01, for control, DEX pretreated, and PSL pretreated cells) were larger in nonpretreated adipocytes than in adipocytes pretreated with glucocorticoids. These results raise the possibility that glucocorticoids, namely, DEX and PSL, stimulate the translocation and subsequent degradative downregulation of PKC, and that this may be pertinent to their inhibitory effects on INS-stimulated glucose transport.

Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization induced by endothelin-1 in cultured canine tracheal smooth muscle cells

Regulation of the increase in inositol 1,4,5-trisphosphate (IP 3) production and intracellular Ca 2+ concentration ([Ca 2+] i) by protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by endothelin-1 (ET-1) led to IP 3 formation and caused an initial transient peak followed by a sustained elevation of [Ca 2+] i in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 μM ) for 30 min blocked the ET-1-induced IP 3 formation and Ca 2+ mobilization. However, this inhibition was reduced after incubating the cells for 8 h with PMA. Following preincubation, ET-1-induced Ca 2+ mobilization recovered with time and reached the same extent of control cells within 48 h. The concentrations of PMA that gave half-maximal inhibition (-IogEC 50) of ET-1-induced IP 3 formation and increase in [Ca 2+] i were 8.6 and 8.4 M, respectively. Prior treatment of TSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate ET-1-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the ET-1-induced IN formation and Ca 2+ mobilization, a change of PKC activity was observed in TSMCs. PMA rapidly decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca 2+] i increase or inhibit independently both responses. The PMA-induced inhibition of responses to ET-1 was associated with an increase in membranous PKC activity.

Down-regulation of protein kinase C attenuates the oxidant hydrogen peroxide-mediated activation of phospholipase A 2 in pulmonary vascular smooth muscle cells

Exposure of rabbit pulmonary vascular smooth muscle cells to H 2O 2 dose-dependently stimulates the cell membrane associated protein kinase C (PKC) activity, phospholipase A 2 (PLA 2) activity, phospholipase A 2 (PLA 2) activity, and arachidonic acid (AA0) release. Pretreatment of the cells with staurosporine (an inhibitor of PKC) prevents AA release and PLA 2 activity caused by H 2O 2. Treatment of the cells with 4β-PMA (an active phorbol ester), or 4α-PMA (an inactive phorbol ester) does not affect basal AA release. In contrast, 4β-PMA significantly stimulates the cell membrane associated PKC activity. Treatment of the cells with 4β-PMA for a short time (upregulation of PKC) augments PLA 2 activity and AA release caused by a sub-optimal dose of H 2O 2 (0.4 mM). Under this condition, staurosporine prevents the stimulatory effects of 4β-PMA on membrane PLA 2 activity, and AA release. In contrast to the up-regulation, pretreatment with 4β-PMA for a longer time (down-regulation of PKC) does not appreciably augment PLA 2 activity and AA release caused by 0.4 mM H 2O 2. Treatment of the cells with an intracellular Ca 2+ antagonist TBM-8 prevents H 2O 2 induced membrane PLA 2 activity and AA release without affecting membrane PKC activity. Treatment of the cells with TMB-8 before addition of 4β-PMA (up-regulation of PKC) followed by incubation with 0.4 mM H 2O 2 does not augment PLA 2 activity and AA release, although membrane PKC activity increases under this condition.

Tobacco smoke tumor promoters, catechol and hydroquinone, induce oxidative regulation of protein kinase C and influence invasion and metastasis of lung carcinoma cells.

Cigarette smoke polyphenolic agents (catechol and hydroquinone) that generate oxidants have been shown to be tumor promoters. Furthermore, oxidants can influence protein kinase C (PKC)-mediated signal transduction. Since terpenoid tumor promoters, phorbol esters, increase invasion and metastasis by activating PKC, we have determined whether polyphenolic agents present in the cigarette smoke condensate (CSC) could also influence these events. Hydroquinone (50 microM), catechol (500 microM), or CSC (50 micrograms/ml) induced an initial cytosol-to-membrane translocation of PKC in LL/2 lung carcinoma cells, followed by a later down-regulation of the enzyme. LL/2 cells treated with these CSC-related agents for a limited time (45 min) and exhibiting high membrane-associated PKC activity, when injected into mice through the tail vein, produced an increase in metastatic nodules in the lungs after 20 days. However, cells treated with CSC-related agents for a prolonged period did not exhibit an increase in metastasis. Agents that decrease the rate of production of reactive oxygen species, such as catalase either alone or in combination with superoxide dismutase, and a cell-permeable iron-chelator, o-phenanthroline, inhibited CSC-mediated membrane association of PKC and metastasis. Prior treatment of CSC with tyrosinase to modify polyphenols resulted in a partial loss of CSC stimulation of metastasis. Furthermore, a cell-permeable Ca2+ chelator and diverse PKC inhibitors, such as calphostin C, hypericin, chelerythrine, and bisindolylmaleimide, inhibited CSC-enhanced metastasis. CSC increased in vitro tumor cell adhesion to endothelial monolayers and to reconstituted basement membrane (Matrigel) and also enhanced the invasion through Matrigel coated on the polycarbonate filters in Transwells. All these CSC effects were found to be temporary and were blocked by the above mentioned antioxidant systems and PKC inhibitors. Thus, these results suggest that the oxidants generated by autooxidation of polyphenolic agents present in tobacco smoke increase tumor cell invasion and metastasis, at least in part by activation of Ca2+/PKC signal transduction. Conceivably, cigarette smoke constituents not only promote tumorigenesis but also may increase the spread of cancer in the body.

Phorbol ester- and retinoic acid-induced regulation of the protein kinase C substrate MARCKS in immortalized hippocampal cells.

The expression of MARCKS, a major protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS protein levels were reduced through an apparent PKC-dependent mechanism. Exposure to 1 microM phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane-associated PKC activity. PKC activity was reduced to < 20% of control values in both soluble and membrane fractions following 1 h of PMA exposure. Significant reductions in MARCKS protein levels were initially observed in membrane and soluble fractions following PMA exposure for 4 and 8 h, respectively. The reduction in MARCKS protein levels was maximal following 24 h of PMA exposure. MARCKS protein expression was also down-regulated in a dose-dependent manner on exposure of HN33 cells to retinoic acid. In cells exposed to 10 microM retinoic acid, the MARCKS protein level was reduced in the membrane fraction within 4 h. Reduction of MARCKS protein levels was maximal (> 90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down-regulation of MARCKS protein in PMA-treated cultures but not in retinoic acid-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyanide induces protein kinase C translocation: blockade by NMDA antagonists.

Activation and translocation of protein kinase C (PKC) during KCN-induced histotoxic hypoxia was studied in rat brain slices prepared from cerebellum, hippocampus, and cortex. Treatment with 1-10 mM KCN produced a significant increase in PKC translocation and enzyme activity in the particulate fraction of cerebellar and hippocampal slices. In cortical slices, PKC activity was not affected by cyanide treatment. The membrane-associated PKC activity reached a maximum 30 minutes after incubation with KCN and remained elevated up to 60 minutes in both the hippocampus and cerebellum. Pretreatment with MK-801 and APV, specific NMDA receptor antagonists, blocked the cyanide-stimulated translocation in the hippocampus and cerebellum, whereas CNQX, an AMPA/kainate receptor antagonist, did not alter the response. These results demonstrate that cyanide stimulates PKC activation and translocation from the cytosol to membranes in select brain areas and NMDA receptor activation mediates this process.

Parathyroid hormone fragments may stimulate bone growth in ovariectomized rats by activating adenylyl cyclase.

PTH is regarded conventionally as a catabolic hormone that stimulates osteoclastic resorption of bone. However, it has been known since 1932 that intermittent pulses of PTH stimulate bone formation in animals and humans. PTH independently activates two signal mechanisms: one that stimulates adenylyl cyclase and one that stimulates protein kinase C (PKC). The goal of this study was to use the 3- to 5-month-old ovariectomized (OVX) rat model to determine which of the two signal mechanisms is responsible for the anabolic action of PTH on bone. OVX triggered a large loss of trabecular bone without significantly affecting the normal slow growth of cortical bone in the distal halves of the femora. Daily injections of human hPTH(1-34) fragment (1 nmol/100 g body weight), which stimulated both adenylyl cyclase and membrane-associated PKC activity in osteoblast-like ROS 17/2 rat osteosarcoma cells, stimulated the growth of both cortical and trabecular bone in the OVX rats. Daily injections of the same dose of hPTH(1-31), which stimulated adenylyl cyclase but not PKC in ROS 17/2 cells, stimulated trabecular bone growth in the OVX rats less effectively than hPTH(1-34), but it stimulated cortical bone growth as rapidly and as dramatically as hPTH(1-34). Injections of equimolar amounts of desamino-hPTH(1-34) [N-propionyl(2-3)hPTH-amide], which stimulated PKC as strongly as hPTH(1-34) in ROS 17/2 cells but had a drastically reduced ability to stimulate adenylyl cyclase, or injections of recombinant hPTH(8-84) which stimulated PKC only in the ROS 17/2 cells, did not stimulate cortical or trabecular bone growth in the OVX animals. Thus, cyclic AMP and cyclic AMP-dependent protein kinases may be the primary mediators of the anabolic action of intermittent pulses of PTH on bone in OVX rats.

The epidermal growth factor mitogenic signal is modulated by protein kinase C in T51B rat liver cells

The regulation of cell proliferation involves a complex interplay between several signal transduction pathways. The effect of EGF on DNA synthesis in serum starved quiescent, synchronized T51B cells was investigated by [ 3H]thymidine incorporation and flow cytometry. 1 nM EGF or readdition of serum initiated G 1 progression and entry into S phase by 18 h and DNA synthesis reached a maximum by 28 h. Low concentrations of EGF markedly stimulated DNA synthesis, but EGF was not as potent as readdition of serum. The effect of EGF on DNA synthesis was only partially blocked by the tyrosine inhibitors genistein and tyrphostin, suggesting that other signalling pathways play a role in EGF-stimulated mitogenesis. 1 nM EGF caused a rapid, transient increase in the activity of membrane-associated protein kinase C (PKC) followed by a longer sustained increase that continued into S phase. TPA (12- O-tetradecanoyl-phorbol-13-acetate) did not mimic EGF, rather it caused a slight stimulation of membrane-associated PKC activity within 1 h followed by a dramatic downregulation of PKC within 4 h. TPA was without effect on DNA synthesis alone, but when added along with EGF or serum TPA caused a significant enhancement of DNA synthesis. Pretreatment of quiescent, serum-deprived T51B cells with TPA reduced the basal level of DNA synthesis; however, under these conditions EGF became as potent a mitogen as serum. We hypothesize that EGF via activation of PKC regulates the activity of its receptor by switching from high affinity to low affinity states. Downregulation of PKC by long term treatment with TPA removes this regulation thus rendering T15B cells more sensitive to exogenous EGF.

Further definition of the protein kinase C activation domain of the parathyroid hormone

The protein kinase C (PKC) activation domain of the parathyroid hormone (PTH) was believed to be the 28-34 region of the molecule. We have now shown that PTH-(29-32) is the smallest PTH fragment that can stimulate significantly membrane-associated PKC activity in ROS 17/2 rat osteosarcoma cells. As was previously shown for full-length PTH-(1-84) and the fully bioactive PTH-(1-34) fragment, there were two peaks in the PKC response to PTH-(29-32): one peak was obtained with low picomolar concentrations and the other with much higher nanomolar concentrations of the fragment. The PKC-activating ability was unaffected by the loss of Asn33 and Phe34, but it was abolished by removing His32. Thus, the PTH-(28-31) and PTH-(29-31) fragments did not stimulate membrane-associated PKC activity. The much larger PTH-(1-31) fragment also did not stimulate membrane-associated PKC activity, although it stimulated adenylyl cyclase as strongly as PTH-(1-34). This functional sensitivity to the loss of the polar His32 was not caused by a specific need for His or another polar amino acid in this position because replacing it with the apolar Leu did not abolish adenylyl cyclase or PKC activation. It is concluded that the minimum, fully functional PKC activation domain of the PTH molecule is Gln29-Asp30-Val31-His32.

Lipopolysaccharide-mediated signal transduction through phospholipase D activation in monocytic cell lines

Lipopolysaccharide (LPS)-induced phospholipase D (PLD) activation was investigated in undifferentiated monocytic leukemic cell lines THP-1 and U-937. Treatment of THP-1 or U-937 cells labelled with [ 32P]orthophosphate, [ 32P]acyl GPC or [ 3H]alkyl GPC with LPS, in the presence of 0.5% ethanol, resulted in the accumulation of labelled phosphatidylethanol (PEt) through PLD activation. LPS-mediated PLD activation of THP-1 or U-937 was inhibited by staurosporine (2 μM) and by protein kinase C (PKC) down-regulation with 12- O-tetradecanoylphorbol 13-acetate (TPA) suggesting a role for PKC. In addition to LPS, TPA, ionomycin and cell-permeant analogs of diacylglycerol also stimulated [ 3H]PEt accumulation. The TPA-induced PEt accumulation was also completely abolished by staurosporine or down-regulation of PKC (> 95% inhibition). Furthermore, the LPS-mediated [ 32P]PEt formation was attenuated by either depletion of extracellular Ca 2+ with EGTA (5 mM) or chelation of intracellular Ca 2+ by BAPTA (30 μM). These results indicate that an increase in intracellular Ca 2+ is necessary for LPS-mediated PLD activation. Further support for PKC activation by LPS was obtained by determining PKC activity in an in vitro assay of histone H1 phosphorylation using [γ- 32P]ATP. In untreated THP-1 cells, approximately 64% of the PKC activity was localized in the cytosol and 36% in the membrane fraction. Treatment of the cells with LPS (10 μg/ml, for 2 h) resulted in an increase of 10% of the membrane-associated PKC activity and a corresponding decrease in the cytosol fraction. These data provide evidence that one of the mechanisms of LPS-mediated signal transduction in human monocytic cell lines involves activation of PLD.

Protein kinase C activity as a potential marker for colorectal neoplasia.

Protein kinase C (PKC) is a mediator of transmembrane signal transduction, important in cell growth and differentiation. Cell activation by extracellular signals is associated with a translocation of PKC from the cytosol to the membrane. We measured and compared PKC activity in cytosol and membrane fractions of normal and neoplastic colorectal tissue. Total and membrane-associated PKC activity in normal colorectal tissue was greater in patients (N = 16) with colorectal cancer compared to that from patients with a normal colonoscopy (N = 16), P < 0.01. A similar trend was noted in PKC activity of normal colorectal tissue from patients with adenomas compared to patients with a normal colonoscopy. PKC activity (total, membrane-associated, percent membrane) was not different in neoplastic colorectal tissue compared to that of adjacent normal tissue. However, there was a considerable range of PKC activity noted in all groups, which would limit the utility of PKC activity as a marker for colorectal neoplasia.

C‐terminal fragments of parathyroid hormone‐related protein, PTHrP‐(107‐111) and (107‐139), and the N‐terminal PTHrP‐(1‐40) fragment stimulate membrane‐associated protein kinase C activity in rat spleen lymphocytes

Membrane-associated protein kinase C (PKC) activity in lymphocytes freshly isolated from rat spleen was stimulated by the C-terminal parathyroid hormone-related protein fragments, PTHrP-(107-111) and PTHrP-(107-139), at concentrations from 10(-3) to 10(4) pM. By contrast, the same concentrations of PTHrP-(120-139), without the 107-111 TRSAW (-Thr-Arg-Ser-Ala-Trp-) sequence of the other C terminal fragments, did not stimulate spleen lymphocyte PKC. Low concentrations of the N-terminal PTHrP-(1-40) fragment also stimulated membrane-associated PKC activity in the spleen lymphocytes. These results suggest that PTHrP might be an important physiological regulator of the immune response.

LONG‐WAVE ULTRAVIOLET RADIATION INDUCES PROTEIN KINASE C IN NORMAL HUMAN KERATINOCYTES

Skin tumor promotion by phorbol ester is believed to be mediated by the phospholipid-dependent ser/thr kinase, protein kinase C (PKC). Long-wave ultraviolet radiation (320-400 nm, UVA), which has also been shown to promote skin tumors, induces elevated levels of PKC in murine fibroblasts, suggesting that UVA may promote the development of basal and squamous cell skin cancers by a mechanism involving PKC. To examine UVA effects on PKC in a model relevant to skin, we maintained normal human epidermal keratinocytes (NHEK) in serum-free medium and exposed the cultured cells to various doses of UVA or to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Fifty minutes after exposure to UVA (5-20 J/cm2), PKC activity was elevated up to three-fold in NHEK cytosolic fractions, and membrane-associated PKC activity was elevated up to two-fold by UVA. The TPA treatment induced a 10-fold increase in membrane-associate PKC activity only. Immunoblot analysis suggested that a UVA-induced increase in PKC protein occurred. Both UVA and TPA reduced the cell number by 50-75% in the first 24-48 h; however, irradiated cultures began to recover at 72 h post-UVA due to an increased proliferative rate beginning after 48 h. Treatment with TPA induced a high level of differentiation as measured by cornified envelope formation. Ultraviolet A irradiation exposure was not followed by increased differentiation. These findings suggest that acute UVA exposure elevates PKC activity in human keratinocytes and may act through PKC to promote actinic skin cancer. The molecular mechanism is like to differ from that of the phorbol esters, however.

The effect of cholecystokinin on intracellular Ca2+, membrane-associated protein kinase-C activity, and progesterone production in chicken granulosa cells.

Nerve fibers immunoreactive for cholecystokinin (CCK) have been observed in the rat ovary, but the function of this gut peptide in the ovary is not known. These studies were designed to investigate the effects of the CCK C-terminal octapeptide (CCK-8) on the intracellular calcium ion concentration ([Ca2+]i), protein kinase-C (PKC) activity, and progesterone secretion in granulosa cells obtained from the two largest preovulatory follicles (F1 and F2) of hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The resting [Ca2+]i in these cells was 96 +/- 5 nM. There was a rapid (i.e. within 5-10 sec) 2- to 4-fold increase in [Ca2+]i in 70% of the cells examined after the addition of 10(-7) M CCK-8. The CCK-8-triggered [Ca2+]i transient was not affected by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA or by pretreating the cells with a Ca2+ channel blocker, such as La3+ (1 mM) or D600 (100 microM). The CCK-8-triggered [Ca2+]i surge was abolished by pretreating the cells with the inhibitor of inositol phospholipid hydrolysis, neomycin (1.5 mM), the CCK antagonists proglumide (1 mM) and benzotript (1 mM), or pertussis toxin (50 ng/ml for 12 h). Incubating granulosa cells with CCK-8 (2 x 10(-7) M) for 10 min stimulated a 1.60 +/- 0.04-fold increase in membrane-associated PKC activity over control levels. In 3-h incubations, CCK-8 (10(-6)-10(-8) M) did not affect basal or LH (20 or 100 ng/ml-stimulated progesterone production. These studies demonstrate that CCK-8 causes a transient increase in chicken granulosa cell [Ca2+]i through the release of Ca2+ from intracellular stores and activates membrane-associated PKC activity, but does not affect progesterone production. These results suggest the presence of G-protein-coupled phospholipase-C-activating CCK receptors on the surface of these cells.

High Membrane-Associated Protein Kinase C Activity Correlates to Tumorigenicity but Not Anchorage-Independence in a Clone of Mouse NIH 3T3 Cells

Anchorage-dependent, nontumorigenic rat F111 fibroblasts have a low level of membrane-associated protein kinase C (PKC) activity. After expression of the polyoma virus middle tumor antigen this activity increased, the cells grew in agar and formed tumors after injection into syngeneic rats or nude mice. Contrary to F111, a clone of mouse NIH 3T3 fibroblasts has a high membrane-associated PKC activity and is as tumorigenic as polyoma-transformed cells, although this clone is still anchorage-dependent. Therefore, membrane-associated PKC activation might be one of the signals leading to tumorigenicity but not necessarily anchorage-independence.

Hydroperoxide-Induced Diacylglycerol Formation and Protein Kinase C Activation in Vascular Endothelial Cells

We examined the effects of hydrogen peroxide (H2O2) and other hydroperoxides on endothelial cell protein kinase C (PKC) activity. Bovine pulmonary artery endothelial cells (BPAEC) were grown to confluent monolayers and PKC activity was determined in an in vitro assay by measuring phosphorylation of H1 histone. In control unstimulated BPAEC, PKC was primarily localized in the cytosol and treatment of BPAEC with H2O2 resulted in a concentration (10−5 M-10−3 M) and time (15 min-60 min.) dependent translocation of the enzyme from the cytosol to the membrane fraction. In addition to H2O2, linoleic acid hydroperoxide treatment of BPAEC also resulted in PKC activation while tert-butyl hydroperoxide and cumene hydroperoxide were not effective. In addition to causing an increase in membrane-associated PKC activity, H2O2 treatment also resulted in the partial loss of cytosolic PKC activity. As diacylglycerol (DAG) is a critical endogenous activator of PKC, we evaluated whether H2O2 exposure resulted in the increased production of DAG. Exposure to 1.0 mM H2O2 resulted in biphasic accumulation of DAG (two- to threefold). The first phase of DAG formation occurred within 5 min of H2O2 exposure while the second phase of accumulation began at 15 min of treatment and plateaued at about 45 min. The metal ions Cu2+ and Fe3+ augmented the H2O2-induced loss of total (cytosolic and particulate) PKC activity in BPAEC. These studies suggest that oxidants modulate PKC activity and increase DAG levels in vascular endothelial cells.

Protein kinase C-activating domains of parathyroid hormone-related protein.

N-terminal fragments of PTH-related protein (PTHrP), PTHrP-(1-34), and PTHrP-(1-40) stimulated both adenylyl cyclase and a mechanism that increases membrane-associated protein kinase C (PKC) activity in ROS 17/2 rat osteosarcoma cells. There were two peaks in the PKC response to the N-terminal PTHrP fragments: one peak was obtained with picomolar and the other with nanomolar PTHrP concentrations. The PKC-stimulating picomolar concentrations of the PTHrP fragments did not detectably stimulate adenylyl cyclase, but the nanomolar concentrations did. Since a similar two-peak response of PKC activity was obtained with PTHrP-(28-34), the single, N-terminal PKC activation domain of the PTHrP is in the same 28-34 region of the molecule as that of PTH despite this region having different primary amino acid sequences in the two hormones. Unlike PTH, PTHrP has a second PKC activation domain, as indicated by the ability of picomolar concentrations of the PTHrP-(107-111) fragment to stimulate maximally membrane-associated PKC activity in the osteosarcoma cells.