Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Addition of transforming growth factor alpha (TGF-alpha) to cultured human keratinocytes results in enhanced expression of TGF-alpha mRNA. This phenomenon of TGF-alpha autoinduction is also observed in a TGF-alpha responsive colon cancer cell line, LIM 1215. In the present study, regulation of TGF-alpha autoinduction is examined in these two cell types. In human keratinocytes, but not in LIM 1215 cells, the increase in steady-state TGF-alpha mRNA following administration of TGF-alpha is due to stabilization of the 4.8-kilobase TGF-alpha transcript, as determined by actinomycin D decay curves. Nuclear run-on experiments confirmed transcriptional control in LIM 1215 cells. Basal and TGF-alpha-stimulated TGF-alpha expression is mediated, at least in part, through a protein kinase C-dependent pathway in both cell types, as determined by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), which attenuates TGF-alpha mRNA accumulation. In the keratinocytes, but not in the LIM 1215 cells, basal TGF-alpha expression is mediated through an epidermal growth factor receptor-dependent pathway, as determined by antibody blockade of the epidermal growth factor receptor. Thus, differential regulation of TGF-alpha autoinduction exists in these nontransformed and transformed epithelial cell types.
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PMID:Differential regulation of transforming growth factor alpha autoinduction in a nontransformed and transformed epithelial cell. 141 97

The relationship between cell differentiation and transforming growth factor alpha (TGF-alpha) expression in human pancreatic cancer cells was analyzed in Capan 1 cells. These cells differentiate either spontaneously or after butyrate treatment. During differentiation (spontaneous or butyrate induced), TGF-alpha messenger RNA (mRNA) levels decreased, whereas the TGF-beta 1 mRNA levels remained unchanged. TGF-alpha was present in cells as proTGF-alpha, which decreased after butyrate treatment. Secretion of TGF-alpha was not found. Under the two conditions of differentiation, the membrane-bound protein kinase C activity was also reduced. Conversely, long-term phorbol ester treatment increased both membrane-bound protein kinase C activity (260%) and TGF-alpha mRNA level (500%), a not significant increase of TGF-beta 1 mRNA was observed. However, phorbol 12-myristate-13-acetate did not induce TGF-alpha synthesis or secretion. These data suggest that expression of TGF-alpha can be reduced in cancer cells; they also suggest the existence of a relationship between TGF-alpha expression and cell differentiation. In addition, the protein kinase C-induced TGF-alpha mRNA level was not followed by the increase of TGF-alpha biosynthesis, suggesting a translational control. Finally, the expression of TGF-alpha and -beta 1 messengers appears to be differently regulated.
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PMID:Decreased expression of transforming growth factor alpha during differentiation of human pancreatic cancer cells. 145 78

Transforming growth factor-alpha (TGF-alpha) is an autocrine growth factor for epidermal keratinocytes that can induce its own expression (autoinduction). Because the regulation of this process may be important for the control of epidermal growth, we examined the roles of EGF receptor tyrosine kinase and protein kinase C (PKC) in TGF-alpha autoinduction in cultured human keratinocytes. Antiphosphotyrosine immunoblot analysis demonstrated that EGF and TGF-alpha rapidly and markedly stimulated tyrosine phosphorylation of a 170 kDa protein in growth factor-deprived keratinocytes. This protein was identified as the EGF receptor by immuno-precipitation using anti-EGF receptor mAbs. Tyrosine phosphorylation and TGF-alpha mRNA accumulation in response to EGF and TGF-alpha were both inhibited by a monoclonal antibody against the EGF receptor and by the EGF receptor tyrosine kinase inhibitor RG50864, demonstrating the involvement of the tyrosine kinase activity of the receptor in TGF-alpha autoinduction. The monoclonal antibody inhibited keratinocyte growth and TGF-alpha autoinduction with similar potency (IC50 approximately 0.1 microgram/ml). TGF-alpha and the PKC activator tetradecanoyl phorbol 12-myristyl, 13-acetate (TPA) had similar effects on TGF-alpha steady-state mRNA levels, suggesting that PKC activation might be a downstream mediator of TGF-alpha autoinduction. However, down-regulation of more than 90% of keratinocyte PKC activity by bryostatin pretreatment abrogated the induction of TGF-alpha mRNA in response to TPA without affecting the autoinductive response or EGF-stimulated tyrosine phosphorylation. These results indicate that EGF receptor and PKC stimulate TGF-alpha gene expression by different pathways, and suggest that PKC is not required for TGF-alpha autoinduction in this system. Moreover, the fact that EGF-stimulated tyrosine phosphorylation and TGF-alpha autoinduction were not potentiated after PKC down-regulation suggests that PKC does not exert a tonic inhibitory influence on EGF receptor tyrosine kinase activity in normal human keratinocytes.
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PMID:Regulation of TGF-alpha expression in human keratinocytes: PKC-dependent and -independent pathways. 157 7

In the present study, we investigated the role of intracellular Ca++ in the stimulation of the Na+/K+/Cl- cotransport in synchronized BALB/c 3T3 cells. The Na+/K+/Cl- cotransport was stimulated by the growth factors EGF, TGF-alpha, IGF-1, and IGF-2, which do not activate protein kinase C, but do induce a transient increase in free cytoplasmic Ca++. In addition, direct activation of protein kinase C by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) did not affect the Na+/K+/Cl- cotransport activity of quiescent cells. The Na+/K+/Cl- cotransport was also stimulated by the above mitogens in cells pretreated with the phorbol ester TPA. This treatment led to a progressive decline in the activity of cellular protein kinase C. This result implies that cells deficient in protein kinase C may still support stimulation of the Na+/K+/Cl- cotransport. Taken as a whole, these findings suggest that the Na+/K+/Cl- cotransport is stimulated predominantly by a protein kinase C-independent mechanism in BALB/c 3T3 fibroblasts. Both the intracellular Ca++ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and two potent calmodulin antagonists, trifluoperazine (TFP) and chloropromazine (CP), blocked serum- and mitogen-stimulated Na+/K+/Cl- cotransport. These results suggest that the Na+/K+/Cl- cotransport is stimulated by an increase of intracellular Ca++ and subsequently by a Ca(++)-calmodulin-mediated pathway in the synchronized BALB/c 3T3 fibroblasts.
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PMID:Na+/K+/Cl- cotransport is stimulated by a Ca(++)-calmodulin-mediated pathway in BALB/c 3T3 fibroblasts. 174 76

Transformation of secondary Sprague-Dawley rat embryo (RE) cells with type 5 adenovirus (Ad5) results in morphologically transformed cells which can undergo a series of sequential changes resulting in enhanced expression of the transformed phenotype, a process termed progression. Selection for a progressed phenotype often occurs after growth in agar or tumor formation in nude mice, and this process is reversible following treatment of cells with 5-azacytidine. In the present study we have analyzed a series of clonal populations of Ad5-transformed RE cells representing different stages in a defined progression lineage. Progression was not associated with alterations in the steady-state levels of mRNA produced by the viral transforming genes, E1A and E1B, or the cellular gene, c-myc. In addition, the tumor-promoting agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which induces expression of a progressed phenotype in Ad5-transformed RE cells, did not significantly alter the RNA transcription rates of the Ad5 E1A or E1B genes, the TPA-inducible gene TPA-S1 or the TPA-responsive genes Pro1 or protein kinase C. TPA did, however, increase by 1 h the steady-state level of c-fos mRNA, but this effect was similar in both progressed and unprogressed cells. Progression also did not involve a change in the RNA transcription rate of a number of cellular and viral genes, including actin, c-Ha-ras, c-myc, v-fos, erbB, TGF-alpha, TGF-beta, Pro-2, transin, TPA-R1, v-myb and c-mos, or other adenovirus genes in addition to E1A and E1B, including E2A and E4. Immunoblotting analysis using E1B polyclonal antiserum further indicated that progression was not associated with changes in the levels of an Mr 21,000 polypeptide encoded by E1B. Similarly, immunoprecipitation analysis with an Ad2 E1A monoclonal antibody indicated similar levels of the Mr 55,000 and 48,000 E1A polypeptides, as well as coprecipitated proteins of Mr 300,000, 107,000 and 105,000 [which is the retinoblastoma (Rb) protein], in E11 and E11-NMT cells. Immunoprecipitation of cell lysates with a monoclonal antibody specific for the Mr 105,000 Rb protein further demonstrated that progression also was not associated with a change in the level or state of phosphorylation of the Rb protein. However, transfection of a human Rb gene (also containing a neomycin resistance gene) into Ad5-transformed RE cells was more inhibitory, with respect to formation of G418-resistant colonies, in unprogressed than in progressed Ad5-transformed RE cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Analysis of viral and cellular gene expression during progression and suppression of the transformed phenotype in type 5 adenovirus-transformed rat embryo cells. 192 6

The ability of staurosporine, a potent inhibitor of protein kinase C, to block certain cellular events initiated by 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) was examined. Treatment of MDA468 breast cancer cells with TPA decreases EGF binding to the cell surface and this effect is blocked by pretreatment with staurosporine with an IC50 of 30 nM. Either 10(-9) M EGF or 100 ng/ml TPA stimulated the accumulation of both EGF receptor and TGF-alpha mRNA and staurosporine (50 nM) completely abolished these mRNA accumulations. Staurosporine did not block EGF-stimulated tyrosine phosphorylation of its receptor as measured by immunoblotting with anti-phosphotyrosine antibodies. The ability of staurosporine to block the mRNA responses of either EGF or TPA suggests that these two agents have common signaling pathways and it implies a role for protein kinase C in the control of EGF receptor and TGF-alpha expression.
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PMID:Inhibition of stimulus-dependent epidermal growth factor receptor and transforming growth factor-alpha mRNA accumulation by the protein kinase C inhibitor staurosporine. 246 87

Normal human epidermal melanocytes were selectively propagated from mixed (keratinocyte-melanocyte) cultures and primary epidermal cell suspensions in serum-free medium, MCDB 153 containing insulin, bovine pituitary extract (BPE), phorbol-12-myristate-13-acetate (PMA), ethanolamine, phosphoethanolamine, and hydrocortisone. Neonatal foreskin melanocytes (NFMs) replicated more readily than adult melanocytes in culture. Early passage NFMs grown in serum-free medium exhibited a population generation time of 24-48 hours. NFMs assumed a less dendritic appearance and were less pigmented than adult melanocytes. PMA or other protein kinase C-activating phorbol esters significantly enhanced mitogenesis of NFMs; however, cAMP-elevating agents were not required for efficient replication of NFMs. Basic fibroblast growth factor (bFGF) was a potent mitogen for NFMs and replaced the requirement for BPE in the culture medium. NFMs expressed a single class of specific, high-affinity receptors for bFGF, exhibiting a Kd = 3 x 10(-11) M and approximately 76,500 receptors/cell. Neither EGF nor TGF-alpha were mitogenic for NFMs, and TGF-beta reversibly inhibited NFM growth. Rapidly growing, early passage NFMs were shown to have cell cycle times of 19.5, 7.5, and 9 hours for G1, S, and G2/M phases of the cell cycle, respectively. Culture of NFMs to confluence or depletion of growth factors from the culture medium caused reversible, G1 phase-specific, cell cycle growth arrest. Senescence of NFMs was associated with irreversible growth arrest in the G1 phase after 40-45 population doublings in culture. Our data demonstrate that basal medium MCDB 153 can be supplemented with defined factors to cultivate selectively two major constituent cell types of the epidermis, the melanocyte and the keratinocyte.
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PMID:Serum-free culture of normal human melanocytes: growth kinetics and growth factor requirements. 255 Apr 77

Transforming growth factor alpha (TGF-alpha) is produced by many transformed cells, but little is known about the regulation of its expression. We examined TGF-alpha mRNA levels in a set of cloned neoplastic cell lines derived by chemical transformation of a normal rat liver epithelial cell. The untransformed parental cell line, WB-344, did not express a detectable level of TGF-alpha mRNA, whereas GP6ac, a transformed line capable of autonomous growth in soft agar, expressed TGF-alpha. When GP6ac cells were treated with agents thought to regulate protein kinase C activity, e.g., the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), TGF-alpha mRNA levels increased by 8- to 11-fold. The induction of TGF-alpha mRNA was detectable at 2 h, was maximal at 8-12 h, and declined by 24 h. Angiotensin, bradykinin, epinephrine, and epidermal growth factor also increased TGF-alpha mRNA by 2- to 5-fold. In contrast, parental WB cells neither expressed TGF-alpha mRNA, nor responded to TPA. TPA also increased epidermal growth factor receptor mRNA in GP6ac cells but the effect was less prolonged; maximal levels were seen at 4 h after TPA exposure and returned to control levels by 12 h. TPA increased TGF-alpha mRNA in GP6ac cells, in part, by increasing transcription of the TGF-alpha gene as measured by run-on transcription rates in isolated nuclei. In addition, the induction of TGF-alpha by TPA was blocked by concurrent incubation with agents that inhibit protein synthesis. However, if TPA was present for at least 2 h, subsequent addition of cycloheximide enhanced the effect of TPA. This indicates that the induction of TGF-alpha in GP6ac cells is comprised of at least two phases demarcated by the requirement for protein synthesis. The time course of induction and the sensitivity to inhibition of protein synthesis distinguish the effect of TPA on TGF-alpha mRNA from that of other genes regulated by TPA, e.g., c-myc and c-fos. These data also suggest that chemical transformation of rat liver epithelial cells leads to expression of TGF-alpha mRNA, and that once expressed, TGF-alpha mRNA can be modulated in a protein kinase C-dependent manner.
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PMID:Regulation of transforming growth factor alpha messenger RNA expression in a chemically transformed rat hepatic epithelial cell line by phorbol ester and hormones. 278 55

Exposure of cultured human epidermal keratinocytes to the protein kinase C (Ca2+- and phospholipid-dependent protein kinase)-activating phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) or 4-beta-phorbol-12,13-didecanoate markedly enhanced accumulation of transforming growth factor-alpha (TGF-alpha) mRNA and secretion of TGF-alpha protein. The nonactivating phorbol ester, 4-alpha-phorbol 12,13-didecanoate, had no effect. In the absence of exogenous growth factors, confluent cultures of keratinocytes express low or undetectable levels of TGF-alpha mRNA and protein. While TPA and epidermal growth factor treatment of keratinocyte cultures deprived of growth factors both induced TGF-alpha mRNA expression, maximum induction by TPA is 5-fold greater than epidermal growth factor. Furthermore, the addition of epidermal growth factor did not enhance TPA-mediated induction of TGF-alpha mRNA expression. Under these experimental conditions, TPA increased levels of secreted TGF-alpha protein by 20-fold at 24 h. Concentration dependence and kinetic studies of TGF-alpha expression showed that TPA (greater than or equal to 1 ng/ml) induced accumulation of TGF-alpha mRNA with an optimum concentration of 10 ng/ml. TGF-alpha mRNA expression increased within 1 h following TPA treatment (10 ng/ml) and peaked at 5 h. At 24 h, TPA-treated cultures still expressed elevated levels of TGF-alpha mRNA (1.7-fold). Protein secretion into the medium was enhanced 2-fold (5 h) to 3-fold (24 h) by TPA treatment of keratinocyte cultures containing growth factors. Prolonged pretreatment (24 h) of keratinocyte cultures with TPA caused marked desensitization of TGF-alpha mRNA expression to repeated stimulation by phorbol ester. The synthetic diacylglycerol, 1,2-sn-dioctanoylglycerol, enhanced levels of TGF-alpha transcription and secretion of TGF-alpha protein. The rate of TGF-alpha mRNA accumulation peaked and declined earlier for 1,2-sn-dioctanoylglycerol compared to TPA. 1,2-sn-Dioctanoylglycerol (50 micrograms/ml) increased production and secretion of TGF-alpha protein, but less than TPA treatment. An inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, also inhibited 1,2-sn-dioctanoylglycerol-mediated accumulation of TGF-alpha mRNA. Cycloheximide failed to inhibit TGF-alpha mRNA expression induced by TPA and, when added alone to keratinocyte cultures, significantly enhanced TGF-alpha mRNA accumulation. Actinomycin D abrogated transcriptional activation of TGF-alpha mRNA by TPA. These studies suggest that activation of protein kinase C by active phorbol esters or diacylglycerols is responsible, at least in part, for TGF-alpha gene expression.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Induction of transforming growth factor-alpha expression in human keratinocytes by phorbol esters. 292 87

The c-erbB-2 gene was first identified by virtue of its cross-hybridization with v-erbB. Nucleotide sequence analysis of complementary DNA clones suggested that the c-erbB-2 gene encodes a growth factor receptor similar to that for EGF. Antibodies against the carboxyl terminal sequence of the c-erbB-2 protein immunoprecipitated a 185-kDa glycoprotein which showed protein-tyrosine kinase activity in vitro. Despite the extensive similarity between the c-erbB-2 protein and EGF receptor, neither EGF nor TGF-alpha bound to the c-erbB-2 protein. Phosphorylation of the c-erbB-2 protein was stimulated by TPA via protein kinase C in vivo. EGF also induced phosphorylation of the c-erbB-2 protein. This phosphorylation occurred not only on serine and threonine residues but also on tyrosine residues. Preliminary data suggested that the latter was mediated by the kinase activity of the EGF receptor. Southern blot analysis of DNAs from primary tumors revealed that the c-erbB-2 gene tends to be amplified in adenocarcinomas, mostly of the stomach and the breast. By screening both human genomic and cDNA libraries using v-yes DNA as a probe, we obtained DNA clones of the c-yes gene, the pseudogene of c-yes, c-fgr gene and c-src gene and two novel yes-related genes, fyn and lyn. Complete nucleotide sequence analysis of the cDNA clones of c-yes, fyn and lyn revealed that these genes encode proteins similar to p60src both in size and sequence.
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PMID:[The erbB-related protooncogenes encoding growth factor receptors]. 349 52


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