Modulation of phospholipase D-mediated phosphatidylglycerol formation by differentiating agents in primary mouse epidermal keratinocytes

The major component of the epidermis, keratinocytes, must continuously proliferate and differentiate to form the mechanical and water permeability barrier of the skin. Our previous data have suggested a potential role in these processes for phospholipase D (PLD), an enzyme that hydrolyzes phospholipids to generate phosphatidic acid. In the presence of primary alcohols, PLD also catalyzes a transphosphatidylation reaction to produce phosphatidylalcohols, and this characteristic has been exploited to monitor the activity of PLD in intact cells. In this report, PLD was demonstrated to utilize the physiological alcohol glycerol to form phosphatidylglycerol (PG) in vitro. In intact primary murine epidermal keratinocytes treated for 24 h with elevated extracellular Ca(2+) levels, but not 1,25-dihydroxyvitamin D(3), incubation with radioactive glycerol resulted in an increase in PLD-mediated radiolabeled PG production. This effect was dose-dependent and biphasic, with maximal PG formation detected after exposure to an intermediate (125 microM) Ca(2+) concentration. Furthermore, the biphasic nature of the response was due, in part, to a corresponding biphasic change in glycerol uptake. Finally, short-term treatment of keratinocytes with phorbol 12-myristate 13-acetate (PMA) failed to increase PG synthesis and inhibited glycerol uptake. Since (1) PMA is reported to activate PLD-1 to a greater extent than PLD-2, (2) 1,25-dihydroxyvitamin D(3) increases the expression/activity of PLD-1 in keratinocytes, and (3) PLD-2 is co-localized with a glycerol channel in keratinocyte membrane microdomains, we speculate that radiolabeled PG production from radioactive glycerol is a measure of PLD-2 activation in these cells. Our results also suggest that PLD-mediated PG synthesis may be regulated at the level of both PLD activity and alcohol substrate availability via changes in glycerol uptake.     

Differential control of proto-oncogene c-myc and c-fos expression in lymphocytes and fibroblasts.

In lymphocytes stimulated with the mitogen phytohaemagglutinin, an inhibitor of the enzyme ADP-ribosyltransferase (ADPRT) completely blocks the proliferative response and the increase in expression of the proto-oncogene c-myc without affecting c-fos significantly. Conversely, in fibroblasts the serum-induced growth is not affected by the ADPRT inhibitor, and both oncogenes are dramatically super-induced. Hence there are differences between lymphocyte and fibroblast early responses to mitogenic stimulation and also between regulation of c-fos and c-myc gene expression.     

An in vivo analysis of c-myc and c-fos expression during terminal erythroid differentiation in mouse spleen progenitors

Using thiamphenicol and scheduled bleeding, we were able to induce an adequate number of erythroid stem cells (CFU-e) in mice in order to conduct an in vivo study of the changing expression of c-myc and c-fos oncogenes during erythropoiesis. Results indicated that c-myc and c-fos are active in erythropoiesis and have a similar pattern of expression. A large decrease in expression of both c-myc and c-fos occurs when erythroid cells begin the biochemical transition into mature phenotypes, i.e., when RNA synthesis is down-regulated.     

Regulation of expression of c-fos and c-myc in rat lymphoma Nb-2 cells

This study examined the effects of the mitogen, prolactin and the cell cycle inhibitors, cyclosporin A and neomycin sulfate, on expression of the proto-oncogenes c-fos and c-myc in the rat lymphoma Nb-2 cell line. Stimulation of quiescent cultures with prolactin resulted in a 2-3-fold increase in the constitutive levels of c-myc mRNA which peaked at 4 h and declined thereafter. c-Fos mRNA was not detected in quiescent or prolactin-stimulated cultures. Cyclosporin A or neomycin sulfate reversibly blocked the mitogenic effect of prolactin on Nb-2 cells, but had little effect on constitutive levels of c-myc. However, the release of Nb-2 cells from a cyclosporin A or a neomycin sulfate block resulted in a rapid transient induction of c-fos which peaked at 0.5-1 h and declined rapidly thereafter. These results indicate that the rapid transient expression of c-fos following release from cell cycle blockage was not sufficient to elicit cell division, but these cells were competent to respond to prolactin. Prolactin allows progression through the cell cycle and enhances c-myc mRNA levels.     

c-fos proto-oncogene expression in the nervous system during mouse development.

I show, by in situ hybridization, that c-fos is expressed in the nervous system during mouse development. This expression was found to be restricted to specific regions at late stages of development (day 16 postcoitum), particularly to the spinal cord, dorsal root ganglia, and olfactory lobe. The c-fos protein may play a role in the maturation of these structures by activating specific genes.     

Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells

Recently, a potent vasoconstrictor peptide, endothelin (EDT), was isolated from vascular endothelial cells. We examined its effect on rat vascular smooth muscle cells (VSMCs). EDT induced the elevation of intracellular calcium, which was dependent on extracellular calcium and inhibited by a calcium-channel antagonist in a competitive manner. EDT caused a rapid and transient increase in the c-fos and c-myc mRNA levels and stimulated the DNA synthesis of VSMCs in a dose-dependent manner. This effect of EDT on the proliferation of VSMCs might be related to the development of atherosclerosis.     

Effect of genetic background on the developmental expression of c-fos and c-myc in chicken

The developmental expression of the protooncogenes, c-fos and c-myc, in muscle and liver of 14-and 19-day embryos and 1-, 6-, 8-and 28-day-old chicks of Athens Canadian Random Bred (ACRB) Single Comb White Leghorn (SCWL) and Peterson X Arbor Acres commercial broiler (PXAA) was determined. For the three stocks of chicken, significant differences were found in c-fos and c-myc expression. For both muscle and liver, averaged across ages, abundance of c-fos RNA was highest in PXAA and lowest in ACRB with differences significant at the P<0.01 level. c-myc RNA levels were significantly higher (P<0.01) in PXAA than in ACRB or SCWL liver. Taken over the developmental period, expression of c-fos RNA in muscle increased at different rates between breeds from 14-day embryo levels to peak levels in 6- to 8-day-old chicks and declined in 28-day-old chicks. Levels of c-fos were much lower in liver and showed no consistent differences related to developmental stage. A steady decline in c-myc from 14-day embryo levels to 28-day-old chicks was found in both muscle and liver. This decline in c-myc levels generally parallels the decline in relative growth rates which occurs in all breeds over the developmental period. In liver, the fast growing PXAA had the highest levels of c-myc. c-fos, on the other hand, showed elevated levels in PXAA for both muscle and liver and distinctly different patterns between these two tissues over the developmental period, suggesting tissue-specific involvement in growth.     

Cell wounding activates phospholipase D in primary mouse keratinocytes

Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D₃, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing.     

c-fos and c-myc expression in human endothelial cells as a function of different culture conditions

Cultured human umbilical vein endothelial cells (HEC) could be induced to express c-fos and c-myc mRNA by either serum or ECGS (endothelial cell growth supplement). Neither agonist separately could support HEC proliferation but the combination did. Expression of c-fos and c-myc mRNA in the presence of both serum and ECGS was similar to that observed after each of the two stimuli was introduced separately. c-fos and c-myc expression in cultured HEC, even if related, is not necessarily accompanied by stimulation of cell growth.     

Neurokinin A induces c-fos, c-jun, and c-myc expression in L6 rat myoblasts

Neurokinin A (NKA), a neuropeptide belonging to the tachykinin family, induced c-fos proto-oncogene mRNA expression in serum-deprived L6J1 rat skeletal myoblasts in vitro. The marked increase reached maximal levels after 15 to 30 min. In contrast to this, c-jun and c-myc proto-oncogene expression were only slightly induced, with peak levels after 30 min. NKA did not stimulate DNA synthesis or cell proliferation in serum-deprived L6J1 myoblasts. We demonstrate a relationship between NKA treatment and induction of c-fos, c-jun and c-myc mRNA expression in serum-deprived L6J1 rat myoblasts. The results on DNA synthesis and cell proliferation indicate that the induced proto-oncogene expression alone is not enough to induce a cellular response to NKA. Possible mechanisms of action are discussed.     

Subcellular distribution of protein kinase C/phorbol ester receptors in differentiating mouse keratinocytes

The activation of protein kinase C (PKC) by diacylglycerol or tumor promoters plays a pivotal role in signal transduction and subsequent activation of cellular processes. Since the activity of this enzyme is dependent on its immediate lipid domain, its relative distribution within the cell may be an important regulatory mechanism. We report here a relative decrease in PKC/phorbol ester receptor associated with the particulate fraction of mouse keratinocytes induced to differentiate by two separate systems. First, proliferating keratinocytes maintained in low Ca2+ (0.09 mM) serum-free medium were induced to differentiate rapidly by the addition of Ca2+ (1.8 mM). A 1.4-fold decrease in the percent of total phorbol receptor binding activity present in the particulate fraction and concomitant increase in binding in the cytosol fraction was evident 20 min after the Ca2+ addition. Second, in keratinocytes that differentiate over a 6 day cultivation period in serum-containing medium with Ca2+ concentration of 1.8 mM, a significant decrease in the percent of the phorbol receptor binding activity present in the particulate fraction was observed as the culture begins to differentiate on days 3 and 4. Maximal phorbol ester binding in the particulate fraction corresponded to the proliferative phase of the culture (day 2), while lower levels of PKC/phorbol ester binding to particulate fractions were noted during the early differentiative phase (days 3 and 4). Addition of the synthetic diacylglycerols 1-oleoyl-2-acetylglycerol or L-alpha-1,2 dioctanyl glycerol at 30 micrograms/ml to proliferating keratinocyte cultures induced a modest increase in two markers of terminal differentiation: cornified envelope formation and transglutaminase levels. These findings, taken together, support the hypothesis that PKC activation plays a role in the initial signalling events for keratinocyte differentiation.     

Protein-coding and non-coding gene expression analysis in differentiating human keratinocytes using a three-dimensional epidermal equivalent

The epidermal compartment is complex and organized into several strata composed of keratinocytes (KCs), including basal, spinous, granular, and cornified layers. The continuous process of self-renewal and barrier formation is dependent on a homeostatic balance achieved amongst KCs involving proliferation, differentiation, and cell death. To determine genes responsible for initiating and maintaining a cornified epidermis, organotypic cultures comprised entirely of stratified KCs creating epidermal equivalents (EE) were raised from a submerged state to an air/liquid (A/L) interface. Compared to the array profile of submerged cultures containing KCs predominantly in a proliferative (relatively undifferentiated) state, EEs raised to an A/L interface displayed a remarkably consistent and distinct profile of mRNAs. Cultures lifted to an A/L interface triggered the induction of gene groups that regulate proliferation, differentiation, and cell death. Next, differentially expressed microRNAs (miRNAs) and long non-coding (lncRNA) RNAs were identified in EEs. Several differentially expressed miRNAs were validated by qRT-PCR and Northern blots. miRNAs 203, 205 and Let-7b were up-regulated at early time points (6, 18 and 24 h) but down-regulated by 120 h. To study the lncRNA regulation in EEs, we profiled lncRNA expression by microarray and validated the results by qRT-PCR. Although the differential expression of several lncRNAs is suggestive of a role in epidermal differentiation, their biological functions remain to be elucidated. The current studies lay the foundation for relevant model systems to address such fundamentally important biological aspects of epidermal structure and function in normal and diseased human skin.     

Induction of c-fos and c-myc expression in T lymphocytes after treatment with recombinant interleukin 1-alpha

Our data demonstrate that an early-acting differentiation/growth factor, such as IL 1, can induce the sequential expression of c-fos and c-myc in lymphocytes. Because such a wide variety of agents induces c-onc expression in lymphocytes, we conclude that the expression of these cellular proto-oncogenes represents a common pathway of response of lymphocytes to an exogenous stimulatory signal regardless of the nature of the factor and its receptor.     

The effects of hydrocortisone on c-fos, c-myc and c-ras oncogene expression in IMR-90 fibroblasts

The effects of hydrocortisone on oncogene expression in human IMR-90 fibroblasts was analyzed by Northern blotting of total RNA. In synchronized fibroblasts stimulated with serum alone, there were two time periods of increased c-fos expression during the G1 phase of the cell cycle. There was no significant difference between cells treated with serum plus hydrocortisone, and cells treated with serum alone with respect to c-fos expression. Quiescent cells showed no change in c-fos expression during the G1 phase of the cell cycle. Three peaks of c-fos expression occur when cells are treated with hydrocortisone alone, but hydrocortisone in the absence of serum is insufficient to initiate DNA synthesis. Hydrocortisone has no effect on c-myc or c-Ha-ras expression in the presence or absence of serum in synchronized fibroblasts. Therefore, the control of mRNA production of the nuclear oncogenes c-fos and c-myc, and the cytoplasmic oncogene c-ras are independent and hydrocortisone may enhance DNA synthesis by increasing c-fos expression.