Tyrosine phosphorylation is an early and specific event involved in primary keratinocyte differentiation.

Very little is known about early molecular events triggering epithelial cell differentiation. We have examined the possible role of tyrosine phosphorylation in this process, as observed in cultures of primary mouse keratinocytes after exposure to calcium or 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunoblotting with phosphotyrosine-specific antibodies as well as direct phosphoamino acid analysis revealed that induction of tyrosine phosphorylation occurs as a very early and specific event in keratinocyte differentiation. Very little or no induction of tyrosine phosphorylation was observed in a keratinocyte cell line resistant to the differentiating effects of calcium. Treatment of cells with tyrosine kinase inhibitors prevented induction of tyrosine phosphorylation by calcium and TPA and interfered with the differentiative effects of these agents. These results suggest that specific activation of tyrosine kinase(s) may play an important regulatory role in keratinocyte differentiation.     

Stimulation of human lymphocytes by phorbol acetate and a calcium ionophore induces an early transient expression of the c-fos oncogene followed by c-myc

After stimulation of human lymphocytes by 12-0 tetradecanoyl phorbol acetate (TPA) and by the calcium ionophore A23187 an early transient expression of proto-oncogene fos followed by an expression of c-myc is observed, as has been described in rodent fibroblast stimulated by growth factors. This observation suggests a direct role of protein Kinase C and of calcium flux an the induction of cellular oncogenes fos and myc that may be associated with the early steps of activation and proliferation of lymphocytes.     

Stimulation and inhibition of growth by EGF in different A431 cell clones is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes

Stimulation of quiescent fibroblasts to growth by polypeptide growth factors is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. In contrast to fibroblasts, A431 cells respond to epidermal growth factor (EGF) with a decreased growth rate. Here we report that, in spite of its growth inhibitory effect, EGF rapidly induces transient expression of c-fos mRNA, followed by the synthesis of nuclear c-fos protein. In addition, EGF treatment resulted in elevated levels of c-myc expression. Practically identical results were obtained with variant A431 clones that are resistant to the inhibitory effect of EGF on cell proliferation. These observations suggest that in A431 cells c-fos and c-myc induction is a primary consequence of growth factor-receptor interaction. Indeed, efficient induction of both genes was also observed with cyanide bromide-cleaved EGF, which has previously been shown to be non-mitogenic but able to trigger early events induced by EGF. We observed strong induction of c-fos and to a lesser extent of c-myc also by TPA, and by the calcium ionophore A23187, indicating an important role for kinase C in proto-oncogene activation by growth factors.     

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc mRNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC8) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC8 it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.     

TRPC channels determine human keratinocyte differentiation: new insight into basal cell carcinoma

Aberrant keratinocyte differentiation is considered to be a key mechanism in the onset of hyperproliferative dermatological diseases, including basal cell carcinoma (BCC). It is, therefore, vital to understand what drives keratinocytes to develop such pathological phenotypes. The role of calcium in keratinocyte differentiation is uncontested but the mechanisms controlling calcium-induced differentiation have yet to be completely elucidated. This study was designed to investigate the role of calcium-permeable TRPC channels in human keratinocyte differentiation and BCC, using a combination of molecular and cell biology approaches, involving electrophysiology and Ca(2+)-imaging, on the HaCaT cell line, primary cultures of normal human keratinocytes, and BCC cells. We demonstrated that TRPC1/TRPC4 channel expression was important for keratinocyte differentiation, as knocking out these channels (by siRNA strategy) prevented the induction of Ca(2+)-induced differentiation. TRPC1/TRPC4-mediated calcium entry and endoplasmic reticulum Ca(2+) content increased significantly in differentiated keratinocytes. However, the failure of BCC cells to differentiate was related to a lack of TRPC channel expression and calcium entry. In summary, our data demonstrate that TRPC1 and TRPC4 channels are key elements in keratinocyte Ca(2+) homeostasis and differentiation and may therefore be responsible for skin pathologies.     

Optimal Differentiation of In Vitro Keratinocytes Requires Multifactorial External Control

For almost 30 years, keratinocyte differentiation has been studied in numerous cell models including keratinocyte primary culture with various supplemented culture media. In this respect, it has become quite difficult to draw comparisons between studies using such a variety of culture conditions. Serum-free condition with low calcium has been used to culture basal proliferating cells, though differentiation is induced by various procedures. These latter include the addition of calcium at mM concentration and a concomitant addition of serum and calcium. Lowering the incubation temperature of cells has also been reported to induce a premature differentiation of keratinocytes in organotypic skin culture. This effect of temperature on keratinocyte differentiation has been poorly depicted, although average human skin temperature has been shown to be about 32°C. However, studying differentiation and quantifying shifts in the differentiation rate of a cell population implies to precisely know i) the proportion of differentiated cells in the whole population, and ii) to which extent and to which level of expression, the induction of a gene or a protein might be considered as a marker of differentiation. This lack has rarely been taken into consideration and has surely led to over-interpretations of single protein induction and to consequent extrapolations to real differentiation processes. By means of paralleled analyses with immunocytofluorescence, flow cytometry, and with multiple differentiation markers quantify by qPCR and western-blot, we studied the paradoxical connection between calcium, serum, multilayer culture and incubation temperature on the differentiation of in vitro keratinocytes. Conversely to previous reports, we have shown that calcium switch is indeed a potent model for inducing calcium-dependent genes, but is not an efficient procedure when one wishes to assess the keratinocyte differentiation rate. Moreover, we have demonstrated that a synergic stimulation by calcium, serum, confluence and lower incubation temperature amplified the differentiation rate.     

Commitment to differentiation and expression of early differentiation markers in murine keratinocytes in vitro are regulated independently of extracellular calcium concentrations

In the epidermis, one of the earliest characterized events in keratinocyte differentiation is the coordinate induction of a pair of keratins specifically expressed in suprabasal cells, keratin 1 (K1) and keratin 10 (K10). Both in vivo and in vitro, extracellular calcium is necessary for several biochemical and structural changes during keratinocyte differentiation. However, it has been unclear if calcium serves as a differentiation signal in keratinocytes. In these studies, expression of suprabasal keratin mRNA and protein is used to test whether the initial differentiation of primary mouse keratinocytes in vitro is dependent on changes in the concentration of extracellular calcium. K1 mRNA was expressed at low levels in cultures of keratinocytes growing on plastic in 0.05 mM calcium but in attached cells was not further induced by increases in the concentration of extracellular calcium. Suspension of the keratinocytes into semi-solid medium induced a rapid and substantial increase in both expression of K1 mRNA and in the percentage of cells expressing suprabasal keratin proteins. The induction was unaffected by the concentration of calcium in the semi-solid medium and could not be enhanced by exposing attached cells to higher calcium before suspension. The induction of K1 mRNA could be inhibited by exposure of the keratinocytes to either EGF or fibronectin. These results suggest that commitment of mouse keratinocytes to terminal differentiation is independent of extracellular calcium and may be regulated primarily by extracellular factors other than calcium.     

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of Ej-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

人膀胱癌中某些癌基因的研究

本实验应用Northern和斑点印渍杂交技术探测了人膀胱癌细胞株中c-myc、c-fos、erbB等癌基因的表达,以及TPA对这些癌基因表达的调控,发现BIU-87细胞有这些癌基因的表达,并能被TPA所增强,同时也发现人膀胱癌组织有c-myc、c-fos、erbB、N-ras基因的高表达。提示蛋白激酶C的激活可以诱导某些癌基因的表达。多种癌基因的表达异常可能在膀胱癌中起重要作用。     

Phenotypic effects of overexpression of PKC beta 1 in rat liver epithelial cells

We have used a previously described retroviral expression vector pMV7-PKC beta 1 to develop derivatives of two rat liver epithelial cell lines, K16 and K22, that stably express about tenfold-higher PKC activity than control cells. Despite these high levels of PKC, these cells did not exhibit gross morphologic changes, anchorage-independent growth, or tumorigenicity. K16PKC-4 and K22PKC-2, two lines with the highest PKC enzyme activity, were studied further in terms of several responses to the phorbol ester tumor promoter TPA. When treated with 100 ng/ml of TPA, the control K16MV7 and K22MV7 cells displayed a slight change in morphology, whereas the K16PKC-4 and K22PKC-2 cells displayed a marked change in morphology. Northern blot analyses demonstrated that TPA induced increased levels of fos, myc, phorbin, and ODC RNAs in control K16MV7 and K22MV7 cells, with maximum induction occurring at about 0.5, 1, 8, and 8 h, respectively. In K16PKC-4 and K22PKC-2 cells, TPA induction of phorbin and ODC RNAs was markedly enhanced, but this was not the case for myc and fos RNAs. In addition, the levels of myc RNA were constitutively higher in both K16PKC-4 and K22PKC-2 cells than in the control cells. Taken together, these results provide direct evidence that PKC plays a critical role in modulating the expression of myc, phorbin, and ODC RNAs. On the other hand, overexpression of PKC beta 1 is not itself sufficient to cause cell transformation.     

c-myc oncogene expression inhibits the initiation of myogenic differentiation

The role of c-myc oncogene expression in myogenic differentiation has been established by transfecting rat myoblasts of the L6 cell line with plasmid pMT-myc, in which the c-myc coding sequences were under the control of the metallothionein I promoter. We observed that the constitutive expression of the exogenous c-myc gene inhibits muscular differentiation. A diminution of the endogenous c-myc gene expression occurs within the first 24 h after the transfer of the cells to a differentiating medium. This early decrease of c-myc expression is required for cell differentiation to occur. We have also observed that exogenous myc gene expression has no effect on endogenous myc expression.     

Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-beta

The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions (1.8 mM Ca2+, differentiation-promoting culture environment) was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF-beta on keratinocyte differentiation is Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there is a clear discrepancy between TGF-beta effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TGF-beta is direct and not induced by differentiation.