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)

The responsiveness of normal human keratinocytes to different modulators of protein kinase C (PKC) was investigated. The PKC agonist TPA, staurosporine (a non-specific inhibitor), and Ro31-8220 (a specific inhibitor) were studied for effect on cell morphology, growth rate, involucrin expression, and intracellular calcium levels. Surprisingly the response to nanomolar concentrations of staurosporine was similar to TPA and induced a fusiform morphology, inhibited growth, increased involucrin levels, and raised intracellular calcium. Staurosporine also increased the number of cornified envelopes, and its action therefore appeared identical to TPA. In contrast, Ro31-8220 had little effect on morphology or growth and blocked both the TPA-induced growth inhibition and calcium rise. Ro31-8220 had no effect on staurosporine-induced growth inhibition but partially reduced its associated calcium rise. These results suggest PKC activation is required for keratinocyte differentiation and that staurosporine acts like a PKC agonist to give a similar effect as TPA. Specific inhibition of PKC by Ro31-8220 inhibits TPA-induced differentiation.
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PMID:Staurosporine, a non-specific PKC inhibitor, induces keratinocyte differentiation and raises intracellular calcium, but Ro31-8220, a specific inhibitor, does not. 751 76

Lysophosphatidic acid (LPA) is a biologically active phospholipid known to have growth factor-like activity on fibroblasts. Although the intracellular signal transduction pathways affected by LPA have been well characterized, the possibility that peptide growth factors are involved in the proliferative response of cells to LPA has not been thoroughly investigated. The focus of this work was to determine the effects of LPA on the proliferation and differentiation of early passage cultured human keratinocytes with emphasis on determining if transforming growth factors (TGF), types alpha and beta, are induced by LPA. The effects of LPA are compared with all-trans-retinoic acid (RA), a structurally unrelated lipid that has previously been shown to induce both TGF alpha and TGF beta and have pronounced effects on keratinocyte proliferation and differentiation. Treatment of cultured human keratinocytes with LPA or RA induced the production of TGF alpha by four- to eightfold. A number of structurally related phospholipids did not mimic the TGF alpha-inducing activity of LPA. LPA is mitogenic for keratinocytes and its stimulatory effect could be blocked with an inhibitory antibody to the EGF/TGF alpha receptor, suggesting that the induction of TGF alpha mediates LPA stimulation of keratinocyte proliferation. LPA and RA also induced both the active and latent forms TGF beta from cultured keratinocytes. Induction of TGF beta may mediate the effects LPA had on keratinocyte differentiation which were apparent by inhibition of proliferation (confluent cultures) and increased involucrin synthesis. Dramatic morphological changes were also observed after LPA treatment. Mechanistic studies suggest that LPA activates both pertussis toxin-sensitive and -insensitive signaling pathways involving protein kinase C activation and protein tyrosine phosphorylation. The effects of LPA on TGF alpha and TGF beta production by keratinocytes likely have in vivo relevance as concluded from rodent studies involving topical LPA treatments.
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PMID:Lysophosphatidic acid induction of transforming growth factors alpha and beta: modulation of proliferation and differentiation in cultured human keratinocytes and mouse skin. 781 33

The altered patterns of expression of gangliosides during density-dependent growth inhibition, oncogenic transformation, and embryogenesis suggest that gangliosides, sialylated membrane glycolipids, may affect cellular proliferation and differentiation. Gangliosides of the "b" pathway of ganglioside synthesis, including GM3, GD3, and GD1b, inhibit the proliferation of cultured keratinocytes without increasing differentiation. We have examined the effect on keratinocyte proliferation and differentiation of supplemental ganglioside GT1b, a more highly sialylated ganglioside of the "b" synthetic pathway that is also present in cultured keratinocytes. In contrast to the lack of effect on differentiation of these other gangliosides, we noted significant induction of keratinocyte differentiation by GT1b, as evidenced by early desmosome formation, and increased cornified envelope formation and expression of involucrin and of the differentiation-specific keratin K1. The addition of GT1b did not cause a shift in intracellular free calcium or alter protein kinase C activity. Alterations in the membrane concentration of ganglioside GT1b, a minor ganglioside component of the keratinocyte membrane, may participate in regulating keratinocyte differentiation.
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PMID:Ganglioside GT1b induces keratinocyte differentiation without activating protein kinase C. 786 10

Thapsigargin raises intracellular free calcium ([Ca2+]i) by potently inhibiting the endoplasmic reticulum Ca-ATPase, which sequesters calcium from the cytosol. In human keratinocytes a rise in [Ca2+]i has been associated with differentiation and therefore we investigated the action of thapsigargin on this process. At concentrations above 3 nM thapsigargin inhibited keratinocyte proliferation. Thapsigargin induced an immediate transient [Ca2+]i rise in calcium-free or 70 microM calcium medium but a more prolonged rise in 2 mM calcium. For keratinocytes cultured in 70 microM calcium medium a late [Ca2+]i rise was also observed, after 6 h, similar to the effect of known differentiation stimuli. However, immunohistochemical techniques did not show any expression of the differentiation-specific protein involucrin, a component of the cornified envelope. When keratinocyte differentiation was induced by an increase in the extracellular calcium from 70 microM to 2 mM abundant involucrin and desmoplakin, a component of desmosomes, were synthesised. Both proteins gave staining patterns which suggested incorporation into structural proteins, but thapsigargin disrupted the calcium-induced pattern of involucrin and desmoplakin synthesis. Thapsigargin did not induce differentiation, possibly due to its inability to activate protein kinase C and raise inositol trisphosphate levels. We conclude that a rise in [Ca2+]i does not alone induce keratinocyte differentiation but may act with other intracellular signals to promote differentiation.
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PMID:Thapsigargin raises intracellular free calcium levels in human keratinocytes and inhibits the coordinated expression of differentiation markers. 826 99

Involucrin is one of the precursor proteins of keratinocyte cornified envelope. Although the formation of the cornified envelope is induced by tumor-promoting phorbol esters, the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the involucrin gene expression remains unknown. We have isolated a 5'-upstream region of human involucrin gene and examined its TPA-dependent promoter activity. The involucrin upstream region with the untranslated first exon was connected to chloramphenicol acetyltransferase (CAT)-involucrin promoter expression vector (INV-CAT) and was transfected into fetal rat keratinizing epidermal (FRSK) cells. The INV-CAT-transfected FRSK cells showed considerable CAT activity that was significantly augmented by the treatment of cells with TPA. FRSK cells that were transfected with a reversely oriented 5'-upstream sequence revealed little CAT activity and did not respond to TPA. The effect of TPA was significantly inhibited by the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7). Other protein kinase C activators (1-oleoyl-2-acetylglycerol and mezerein) also induced the INV-CAT promoter activity, whereas 4-O-methyl phorbol myristate acetate, a very weak protein kinase C activator, had only a slight effect. Analysis of the nucleotide sequence of the 5'-upstream region detected several 5'-TGANTCAA-3' sequences that are highly conserved TPA-response elements (TRE). Cotransfection of both c-jun and c-fos expression vectors with the INV-CAT vector into FRSK cells resulted in increased CAT activity. Cotransfection of either the c-jun or c-fos vector singly with the INV-CAT vector into FRSK cells had negligible effects. Dexamethasone significantly inhibited the TPA-induced promoter activity in the INV-CAT-transfected FRSK cells. These results indicate that involucrin gene expression is positively controlled by TPA through the activation of the protein kinase C/TRE system.
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PMID:Analysis of the 5'-upstream promoter region of human involucrin gene: activation by 12-O-tetradecanoylphorbol-13-acetate. 838 Aug 29

The epidermis is a tissue that undergoes a very complex and tightly controlled differentiation program. The elaboration of this program is generally flawless, resulting in the production of an effective protective barrier for the organism. Many of the genes expressed during keratinocyte differentiation are expressed in a coordinate manner; this suggests that common regulatory models may emerge. The simplest model envisions a 'common regulatory element' that is possessed by all genes that are regulated together (e.g., involucrin and transglutaminase type 1). Studies to date, however, have not identified any such elements and, if anything, the available studies suggest that appropriate expression of each gene is achieved using sometime subtly and sometime grossly different mechanisms. Recent studies indicate that a variety of transcription factors (AP1, AP2, POU domain. Sp1, STAT factors) are expressed in the epidermis and, in many cases, multiple members of several families are present (e.g., AP1 and POU domain factors). The simultaneous expression of multiple members of a single transcription factor family provides numerous opportunities for complex regulation. Some studies suggest that specific members of these families interact with specific keratinocyte genes. The best studied of these families in epidermis is the AP1 family of factors. All of the known AP1 factors are expressed in epidermis [52] and each is expressed in a specific spatial pattern that suggests the potential to regulate multiple genes. It will be important to determine the role of each of these members in regulating keratinocyte gene expression. Finally, information is beginning to emerge regarding signal transduction in keratinocytes. Some of the early events in signal transduction have been identified (e.g., PLC and PKC activation, etc.) and some of the molecular targets of these pathways (e.g., AP1 transcription factors) are beginning to be identified. Eventually we can expect to elucidation of all of the steps between the interaction of the stimulating agent with its receptor and the activation of target gene expression.
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PMID:Transcription factor regulation of epidermal keratinocyte gene expression. 898 19

Different chemicals that specifically and selectively inhibit or activate protein kinases have been used to define the possible roles of these enzymes in the different steps of epidermal differentiation. Using HaCaT keratinocytes as a model, and under conditions in which cell proliferation is minimally affected, we found that tyrosine kinase inhibition leads to an inhibition of early (spinous; keratin k10 expression) and late (granulosum; involucrin expression) differentiation processes. cGMP- and cAMP-dependent protein kinases appear to modulate the transition from spinous to granular differentiation, a process which seems to be negatively controlled by protein phosphatases. Finally, enzymes belonging to the protein kinase C family appear to facilitate the transition from spinous to granular differentiation programmes while inhibiting the early steps of epidermal differentiation.
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PMID:Role of protein kinases in the in vitro differentiation of human epidermal HaCaT cells. 927 24

Cell lines are useful as models if they retain the relevant characteristics of the tissue of origin. We compared two human squamous carcinoma cell lines derived from tumors of the tongue that vary in their extent of differentiation, with human biopsies of carcinomas of the tongue that were either poorly or well-differentiated. The mRNA levels of suprabasal cell proteins (keratin K13, involucrin, transglutaminase) and of protein kinase C (PKC) isozymes were measured by RT-PCR. Apart from PKC beta and PKC delta (mostly expressed by Langerhans cells and missing in culture), qualitatively similar patterns were found in vitro and in vivo. The more differentiated cells had expression levels moderately lower to higher than the normal controls. The poorly differentiated cells generally had substantially lower levels.
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PMID:Differentiation markers in oral carcinoma cell lines and tumors. 949 76

Involucrin is one of the precursor proteins of the cornified cell envelope that is formed beneath the cell membrane during terminal differentiation of keratinocytes. 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a potent protein kinase C (PKC) activator, induces terminal differentiation of keratinocytes. We previously demonstrated that involucrin promoter activity is stimulated by TPA in cultured fetal rat skin keratinocytes. PKC is a large family of proteins and keratinocytes containing five PKC isozymes: alpha, delta, epsilon, eta, and zeta. In order to determine the role of the PKC isozyme(s) on involucrin gene expression, we constructed the chloramphenicol acetyl transferase (CAT)-involucrin promoter expression vector by connecting the 5'-upstream region of the human involucrin gene containing the untranslated first exon to the CAT reporter gene. The CAT-involucrin promoter expression vector was transfected with various PKC isozyme expression vectors into SV40-transformed human keratinocytes (SVHK cells). Transfection of the CAT-involucrin promoter expression vector with PKC-alpha or PKC-eta expression vectors resulted in a significant increase in the TPA-dependent involucrin promoter activity. The PKC inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride, inhibited the promoter activity stimulated by TPA. Transfection of PKC-delta, -epsilon, and -zeta had no effect on the involucrin-promoter activity. Although the promoter activity was stimulated by transfection of PKC-gamma, TPA did not enhance the promoter activity in the PKC-gamma-transfected SVHK cells. Previously we showed three AP-1 binding sites (AP1-1, -2, and -3) on the involucrin promoter region. Both the basal and the TPA-stimulated involucrin promoter activities were suppressed by deleting the AP1-1 site (-119 to -113) that is the most proximal to the transcription start site. The deletion of AP1-2 (-297 to -303) or AP1-3 (-447 to -453) did not affect the involucrin promoter activity. Gel retardation analyses disclosed that TPA stimulated the specific DNA binding of the nuclear protein(s) of control, PKC-alpha, or PKC-eta-transfected SVHK cells, but not of PKC-gamma-transfected cells. Addition of anti-c-Jun and anti-c-Fos antibodies decreased the specific protein-DNA complex band with a concomitant appearance of supershifted bands. These results indicate that PKC, specifically PKC-alpha and PKC-eta, mediates the TPA-dependent activation of involucrin gene expression of SVHK cells. PKC-gamma, which is not present in keratinocytes, also induces involucrin gene expression in a TPA-independent manner, when introduced into SVHK cells.
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PMID:The alpha and eta isoforms of protein kinase C stimulate transcription of human involucrin gene. 950 39

Involucrin is a marker of keratinocyte terminal differentiation. Our previous studies show that involucrin mRNA levels are increased by the keratinocyte differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Welter, J. F., Crish, J. F., Agarwal, C., and Eckert, R. L. (1995) J. Biol. Chem. 270, 12614-12622). We now study the signaling cascade responsible for this regulation. Protein kinase C and tyrosine kinase inhibitors inhibit both the TPA-dependent mRNA increase and the TPA-dependent increase in hINV promoter activity. The relevant response element is located within the promoter proximal regulatory region and includes an AP1 site, AP1-1. Co-transfection of the hINV promoter with dominant negative forms of Ras, MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun inhibit the TPA-dependent increase. Wild type MEKK1 enhances promoter activity and the activity can be inhibited by dominant negative MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun. In contrast, wild type Raf-1, ERK1, ERK2, MEK4, or JNK1 produced no change in activity and the dominant negative forms of these kinases failed to suppress TPA-dependent transcription. Treatment with an S6 kinase (S6K) inhibitor, or transfection with constitutively active S6K produced relatively minor changes in promoter activity, ruling out a regulatory role for S6K. These results suggest that activation of involucrin transcription involves a pathway that includes protein kinase C, Ras, MEKK1, MEK3, and p38/RK. Additional pathways that transfer MEKK1 activation via MEK1 and MEK7 also may function, but the downstream targets of these kinases need to be identified. AP1 transcription factors appear to be the ultimate target of this regulation.
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PMID:Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway. 973 28


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