Article |
Address correspondence to Sergei A. Grando, Dept. of Dermatology, University of California, Davis, UC Davis Medical Center, 4860 Y Street, #3400, Sacramento, CA 95817. Tel.: (916) 734-6057. Fax: (916) 734-6793. E-mail: sagrando{at}ucdavis.edu
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Abstract |
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Key Words: cell cycle; differentiation; 7 acetylcholine receptor; epidermis; knockout mouse
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Introduction |
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ACh is a ubiquitous chemical in life that, although is best known for its role in neurotransmission, is produced by practically all types of live cells and is remarkably abundant in the epidermis and other types of the surface epithelium (Grando et al., 1993b; Wessler et al., 1999). It has become evident that ACh can regulate tissue homeostasis in an autocrine and paracrine fashions by exhibiting a plethora of biological effects on different cell types (Wessler et al., 1998). The level of free tissue ACh is controlled by the cholinergic enzymes choline acetyltransferase and acetylcholinesterase that are present in nonneuronal cells lining the cutaneous, respiratory and alimentary tracts, and blood vessels. In these nonneuronal locations, ACh signaling can be mediated by muscarinic and nicotinic receptors. Binding of ACh to the cell membrane receptors elicits several diverse and simultaneous biochemical events, the "biological sum" of which, together with cumulative effects of other hormonal and environmental stimuli, determines a distinct change in the cell cycle.
The nAChRs are classic representatives of the Cys loop superfamily of ligand-gated ion channel proteins or ionotropic receptors, mediating the influx of Na+ and Ca2+ and efflux of K+ (Steinbach, 1990). The differences in subunit composition of nAChRs determine the functional and pharmacological characteristics of the ion channels formed. 12 nAChR subunit genes encoding a pentameric protein have been identified and designated 2
10 and three ß2ß4, and each subunit has four putative transmembrane-spanning domains (M1M4) and a similar topological structure. Each of
7,
8, and
9 subunits is capable of forming functional homomeric nAChR channels, which are
-bungarotoxin (
-BTX) sensitive. RT-PCR has amplified
3,
5,
7,
9,
10, ß2, and ß4 subunits from human keratinocytes (Grando et al., 1995, 1996; Nguyen et al., 2000a, 2001; Sgard et al., 2002), indicating that keratinocytes express both heteromeric and homomeric nAChR channels on their cell membrane. The differences in subunit composition of nAChRs determine the functional and pharmacological characteristics of the ion channels formed.
Current research results indicate that biological effects of ACh in the skin are finely tuned to regulation of each phase of the cell cycle via the intracellular signaling pathways coupled by each particular type of nAChRs (Grando, 1997, 2001). In keratinocytes, nAChRs control cell viability, proliferation, differentiation, adhesion, and motility, and constant stimulation of keratinocyte nAChRs with endogenously secreted ACh produced by these cells is essential for cell survival. We have demonstrated recently that programmed cell death of keratinocytes culminates in apoptotic secretion of a humectant upon secretagogue action of ACh and that activation of ACh signaling through the 7 nAChR, which is predominantly expressed by mature keratinocytes, is essential for a sustained turnover of the epidermis in humans (Nguyen et al., 2001).
This study was designed to ultimately determine the role for 7 nAChR in mediating physiologic control of keratinocyte differentiation by endogenous ACh. Alterations in the nicotinergic regulation of keratinocyte cycle progression, differentiation, and apoptosis were investigated in three independent models of functionally inactivated
7 nAChR: in cultured human keratinocytes treated with
-BTX or antisense oligonucleotides (AsOs) and epidermal keratinocytes grown from and residing in the skin of KO mice with homozygous-null mutation of the gene-encoding
7 nAChR subunit. We found that pharmacological blockage of
7 nAChR with
-BTX inhibits nicotine (Nic)-induced influx of 45Ca2+ in human keratinocytes, which is associated with an inhibition of Nic-induced terminal differentiation of these cells. Functional inactivation of
7 nAChRs in cultured human keratinocytes with AsOs abolished high extracellular Ca2+-induced up-regulated synthesis of the terminal differentiation proteins. Terminal differentiation gene expression was found to be down-regulated in the epidermis of
7 KO mice whose keratinocytes demonstrated profound alterations in the normal cell cycle progression and apoptosis when grown in culture. The
7-/- keratinocytes also demonstrated changes in the gene expression of
3,
5,
9, and
10 nAChR subunits, suggesting that ACh signaling in these cells is rerouted to alternative nicotinergic pathways.
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Results |
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Abnormal keratinocyte differentiation in the skin of 7 KO mice
To correlate changes in the cell cycle and differentiation gene expression resulting from inactivation of 7 nAChR-coupled signaling pathways in vitro with the in vivo phenotype caused by the absence
7 nAChR channels in the epidermis, we studied pups delivered by
7+/- mice, followed by genotyping (Fig. 4 A). Compared with wild-type
7+/+ mice aged from 1 to 3 wk, whose epidermis usually consists of one to two rows of live nucleated keratinocytes and a compact horny layer comprised of dead corneocytes (Fig. 4 B),
7-/- mice featured thickened, multilayered epidermis (Fig. 4 C). In addition to the lowermost basal layer, the epidermis in
7-/- mice contained an additional two to three suprabasilar rows of pale and enlarged keratinocytes and from one to three rows of granular keratinocytes located just below widened and loose horny layer. Thus, the phenotypic abnormalities in the epidermis of
7 KO mice were consistent with retention hyperkeratosis, which is a morphologic manifestation of delayed epidermal turnover.
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Findings of down-regulated expression of terminal differentiation genes in keratinocytes residing in the epidermis of 7-/- mice were corroborated by results of the Western blot assay (Fig. 4 E). The
7 deletion was associated with the decrease of the filaggrin and cytokeratin 1 and 10 proteins.
To ultimately determine changes in the differentiation proteins in epidermis of 7 KO mice, we measured the relative intensities of specific staining of keratinocytes produced by antibodies against the keratohyaline proteins filaggrin and loricrin and the intermediate filament proteins cytokeratin 1 and 6, using semiquantitative immunofluorescence (IF) assay. We found that in the epidermis of
7-/- mice, the abundance of terminally differentiated keratinocytes expressing filaggrin, loricrin, and cytokeratin 1 was significantly (P < 0.05) less than that in the epidermis of
7+/+ mice (Fig. 4 F). In marked contrast, the intensity of epidermal staining for cytokeratin 6, a marker of rapidly proliferating, immature keratinocytes (Foley et al., 1998; Gibbs et al., 2000), was significantly increased (P < 0.05), which is consistent with the appearance of the prolonged epidermal turnover phenotype in
7-/- mice.
Abnormalities in cell cycle regulation of 7 KO keratinocytes
When cell cycle and apoptosis gene expression in 7-/- keratinocytes was analyzed by RT-PCR, we found that Ki-67, cyclin D1, and PCNA were increased by 52, 77, and 52%, respectively, compared with
7+/+ cells (Fig. 5 A). The mRNA level of p53 also increased by 54%. By immunoblotting, we found that the relative amount of Ki-67, cyclin D1, PCNA, and p53 were increased in
7-/- keratinocytes (Fig. 5 B). On the other hand, the mRNA and protein levels of caspase-3 decreased 24 and 57%, respectively, whereas those of Bcl-2 both increased (Fig. 5).
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Altered expressions of nicotinic receptor subunits in 7 KO keratinocytes
To test a hypothesis that mutational deletion of Acra7 in keratinocytes evokes changes in the relative amounts of different nAChR channels, we investigated expression of the genes coding for 3,
5,
9, and
10 subunits in
7-/- versus
7+/+ keratinocytes. By RT-PCR, we found that the expression of the gene coding for
3 in
7-/- keratinocytes was up-regulated by 56%, whereas that of
5 was apparently unchanged (Fig. 6 A). Results of the Western blotting assay showed an 86% increase of the relative amount of
3 protein in
7-/- keratinocytes (Fig. 6 B). The protein level of
5 was found to be unchanged. These results indicated that although the total number of
3 containing nAChR increases in the epidermis of
7 KO mice, the proportion of the
3 nAChRs containing
5 subunit is actually less then in wild-type mice. The relative amounts of both mRNA and proteins of
9 and
10 subunits were elevated in
7-/- keratinocytes (Fig. 6). However, an increase of the protein level of
9 by 63% exceeded that of
10 subunit, indicating that both the heteromeric
9
10 and the homomeric
9-made ACh-gated ion channels were up-regulated in
7-/- keratinocytes.
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Discussion |
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Findings of the new and important biological function of "neuronal" 7 nAChR in a nonneuronal location such as the physiologic control of homeostasis and terminal differentiation of the stratified squamous epithelium was anticipated based on the following reasons. First, despite multiple morphological, biochemical, and electrophysiological studies, the functions of neuronal
-BTX binding sites in the mammalian brain remain largely unknown. Furthermore, a mutation deleting the last three exons of the gene for the
7 nAChR subunit that completely eliminates its potential for participation in an ion channel does not alter normal general appearance, growth, survival, gait, anatomy, and baseline behavioral responses (Orr-Urtreger et al., 1997; Paylor et al., 1998). Thus, although the Acra7 homozygous mutant mice demonstrated that the
7 subunit is not essential for normal development or for apparently normal neurological function, they proved to have phenotypic abnormalities in the epidermis, thus providing a valuable tool for defining the functional role of the keratinocyte
7 nAChR channel in the epidermis.
Second, in addition to modulation of neurotransmitter release the 7 nAChR has been implicated in regulating neuronal growth and differentiation via a large variety of genomic and nongenomic effects, including the promotion of neuronal proliferation (Quik et al., 1994; Plummer et al., 2000), neuroprotection (Gueorguiev et al., 2000; Li et al., 2000; Garrido et al., 2001), and induction of apoptosis (Renshaw et al., 1993; Hory-Lee and Frank, 1995; Berger et al., 1998). Neuronal
7 nAChR acts through different intracellular transduction pathways to protect or kill cells (Li et al., 1999). It has been proposed that
7 nAChR helps regulate neuronal development by modulating intracellular Ca2+ levels and thus affecting neuronal differentiation and synaptogenesis (Broide and Leslie, 1999). The genomic effects downstream of
7 nAChR are represented by activation of tyrosine hydroxylase and dopamine ß-hydroxylase gene expression in PC12 cells (Gueorguiev et al., 2000), whereas the nongenomic pathways involve regulation of protein phosphorylation (Schuller et al., 2000; Kihara et al., 2001).
Third, the 7 subunit is abundantly expressed in the epithelial cells lining skin, oral mucosa, esophagus, trachea, and bronchi, in which nicotinergic stimulation alters cellular metabolism of Ca2+ (Grando et al., 1996; Zia et al., 1997; Nguyen et al., 2000a), endothelial cells (Wang et al., 2001), and in cells surrounding large airways and blood vessels, alveolar type II cells, free alveolar macrophages, and pulmonary neuroendocrine cells (Sekhon et al., 1999). In the mammalian fetal lung,
7 nAChR may regulate neuropeptide release, collagen expression, and ultimately lung development (Sekhon et al., 1999). The expression of
7 nAChR channels on the cell membrane of nonneuronal cells is modulated by exposure to Nic (Zia et al., 1997; Arredondo et al., 2001), which may provide a mechanism for Nic-induced changes in gene expression (Arredondo et al., 2001; Zhang et al., 2001a, b), proliferation (Waggoner and Wang, 1994; Stone et al., 2001), apoptosis (LeSage et al., 1999; Heeschen et al., 2001), secretion (LeSage et al., 1999), and tumor growth (Heeschen et al., 2001) in nonneuronal locations.
The contribution of different nAChR subunits to formation of ACh-gated nicotinic ion channels in the plasma membrane of keratinocytes changes with keratinocyte maturation (Zia et al., 2000). Antibody mapping studies in human epidermis have shown that the bulk of 7 immunoreactivity is localized to the cell membranes of mature keratinocytes comprising the granular layer (Nguyen et al., 2001). In keratinocyte cultures, the abundant expression of
7 was observed on the cell membrane of mature cells, which required preincubation of cultures in KGM containing a differentiation-inducing concentration of Ca2+ or Nic (Zia et al., 2000). In contrast, the
3-containing nAChRs are present at the earliest stages of keratinocyte development (Nguyen et al., 2000a; Zia et al., 2000). Extracellular Ca2+ has been shown to regulate responses of both
3- and
7-containing nAChRs on chick ciliary ganglion neurons (Liu and Berg, 1999). Although both
3 and
7 subunits can contribute to the nAChRs that are permeable to Ca2+, the ACh-gated ion channels composed of the
7 subunits have the greatest Ca2+ permeability (Seguela et al., 1993). The results of this study demonstrated that the need to preincubate keratinocytes at differentiation-inducing concentrations of extracellular Ca2+ in order to increase the sensitivity of their response to Nic to a blockage with
-BTX in the 45Ca2+ influx assay is explained by up-regulated expression of
7 nAChRs.
Results of this study demonstrate that ACh signaling through 7 nAChR channels controls the maturation and the cornification stages of keratinocyte development in the epidermis. Downstream signaling from
7 nAChR regulates expression of cell cycle progression, apoptosis, and terminal differentiation regulators at the transcriptional and/or translational levels. These effects may be mediated, at least in part, by changes in Ca2+ metabolism. A "gain of function" mutation of the ACh-gated ion channels comprised by
7 subunits demonstrated that neurons expressing only mutant nAChRs are susceptible to abnormal apoptosis and degeneration, possibly due to increased Ca2+ influx (Treinin and Chalfie, 1995; Orr-Urtreger et al., 2000; Broide et al., 2001). We found that neither
-BTX could completely block Nic-induced differential of keratinocytes nor anti-
7 AsOs could completely abolish the process of cornification elicited by increasing the concentration of extracellular Ca2+ in KGM. Instead of using Nic to induce keratinocytes differentiation as in experiments with
-BTX, the differentiation of anti-
7 AsOs-treated keratinocytes was induced through alternative pathway(s) sensitive to high extracellular Ca2+, since in these cells the
7 nAChR pathway was inactivated due to treatment with anti-
7 AsOs. These findings suggest that the
7 nAChR-mediated pathway works together with other cholinergic and noncholinergic signaling pathways to sustain a constant advancement of a keratinocyte through its differentiation stages toward its programmed death. In acute experiments, such as treatment of cells with
-BTX or anti-
7 AsOs, the alternative pathway apparently could not get engaged fast enough to compensate for the missing function, which is illustrated by an approximately fivefold drop in the number of cells capable of spontaneous cornified envelope formation (Fig. 1). In marked contrast, in the epidermis of
7 KO mice the process of cornification, although delayed, proceeds via a normal path, surfacing skin of these mice with an impermeable barrier or the stratum corneum. Therefore, a lesser magnitude of changes of the gene expression in keratinocytes residing in the epidermis of
7 KO mice (Fig. 4 E) compared with keratinocytes treated with anti-
7 AsOs (Fig. 3 C), as judged from the results of the Western blotting assay, may be explained by putative physiologic backup mechanisms activated during the development of a KO mouse but lacking in the cells treated with AsOs in which the
7 AChRs are inactivated acutely at the posttranscriptional level.
To test a hypothesis that mutational deletion of 7 brings about changes in the repertoire of nAChR channels, we determined the ratios of different
subunit gene expression in
7-/- keratinocytes. We found alterations in the expression of
3,
9, and
10 nAChRs subunits, indicating that the nicotinergic signaling in the skin of
7 KO mice is predominantly mediated via a nAChR complex containing
3 without
5 and both homomeric
9- and heteromeric
9
10-made nAChRs. This switch in subunit composition of the nAChR-gated ion channels may, in turn, bring about a corresponding switch in the ionic properties of the ion channels formed because of shifting of the nicotinergic signaling to the nAChRs that differ in subunit composition, pharmacology, conductance, and kinetics and in their permeability to and modulation by Ca2+. For instance, it has been shown that
5 subunit increases Ca2+ permeability of
3 nAChR so that the Ca2+ permeability of
3ß2
5 nAChRs is comparable to that of
7 nAChRs (Gerzanich et al., 1998). Hence, a relative decrease of the proportion of
3 nAChRs containing
5 subunits, i.e.,
3ß2
5, in
7-/- keratinocytes can bring about corresponding changes in the ionic properties of the channel, leading to a complex changes in cell cycle regulation, including proliferation-inducing effects, DNA repair and replication anomalies, and antiapoptotic gene activation. On the other hand, up-regulated expression of
9-containing nAChRs that mediate proapoptotic action of ACh at the granular cellcorneocyte transition, which culminates in programmed cell death within the epidermis (Nguyen et al., 2001), may compensate for a lacking component of the nicotinergic control of terminal differentiation of
7-/- keratinocytes, allowing formation of the functional epidermal barrier in
7 KO mice. The nAChR incorporating
9 subunits represents a novel ionotropic and metabotropic receptor/Ca2+ channel (Elgoyhen et al., 1994; Glowatzki et al., 1995; Wikstrom et al., 1998). Compared with homomeric
9 channels, the
9
10 nAChR channel displays faster and more extensive agonist-mediated desensitization, a distinct current-voltage relationship, and a biphasic response to changes in extracellular Ca2+ ions (Elgoyhen et al., 2001). Thus, although the use of KO mice is probably the most straightforward and rewarding approach to dissect biological function of each particular type of keratinocyte nAChRs, providing an unambiguous mechanistic insight into differential control of keratinocyte functions by ACh, the missing function may be partially compensated or obscured due to engagement of the alternative regulatory pathways.
In conclusion, the comprehensive analysis of the biological role of 7 nAChR in keratinocytes revealed its important role in sustaining normal unfolding of the genetically determined program of cell differentiation eventuating in cell death, or cornification, which is required for formation of the skin barrier. The ACh signaling through
7-made channels may evoke rapid and profound changes in the cellular metabolism of free Ca2+ due to modulation of its transmembrane flux. The downstream signaling apparently harbor both genomic and nongenomic effects, the biologic sum of which determines the rate of keratinocyte progression through the differential steps. In Acra7 homozygous mutant mice, the missing regulatory pathway causes transient changes in skin phenotype characteristic of delayed epidermal turnover. The changes are partially compensated due to redirection of the nicotinergic signaling via the
3-type keratinocyte nAChRs that in the past were found to be associated with immature cell phenotype (Zia et al., 2000), and the
9-type keratinocyte nAChRs that are coupled to regulation of keratinocyte apoptotic secretion (Nguyen et al., 2001).
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Materials and methods |
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7 KO mice and murine keratinocyte cultures
The 7 KO mice used in experiments were Acra7-deficient (
7 null) mice generated as described previously (Orr-Urtreger et al., 1997). All control mice were
7+/+ littermates of
7+/- mice. The animals were killed, and skin samples were collected. The samples destined to RNA and protein extractions were fresh-frozen in liquid nitrogen or freshly embedded in the OCT Tissue Tek compound (Sakura) for use in IF experiments. All of the experiments were conducted by an experimenter that was blind to the genotype of the mice. The genotyping was performed by PCR and Southern analysis as detailed elsewhere (Orr-Urtreger et al., 1997). Cell cultures were grown at 37°C and 5% CO2 in 25 cm2 Falcon culture flasks using the cell culture techniques optimized for mouse keratinocytes (Li et al., 1995; Lee et al., 1997).
Immunocytochemical assay
Immunocytochemical analysis of nicotinergic effects on the expression of differentiation markers was performed in situ in keratinocyte monolayers as described previously (Grando et al., 1996). Stained monolayers were examined microscopically and photographed. The numbers of cytokeratin 10, transglutaminase-, involucrin-, and filaggrin-positive cells were counted in at least three different microscopic fields at the magnification x200, and the results were expressed as a percentage of the total cells. At least 50 cell per each microscopic field were examined.
IF assay
The IF experiments with skin samples of 7-/- and
7+/+ mice were performed as detailed previously (Ndoye et al., 1998) using a computer-assisted image analysis with a software package purchased from Scanalytics. The intensity of fluorescence was calculated pixel by pixel by dividing the summation of the fluorescence intensity of all pixels by the area occupied by the pixels (i.e., segment) and then subtracting the mean intensity of fluorescence of a tissue-free segment (i.e., background).
45Ca2+ influx assay
The experiments were performed in triplicate samples according to our modification (Zia et al., 2000) of standard protocols (De Aizpurua et al., 1988). Briefly, freshly isolated human neonatal foreskin keratinocytes were counted with a hemocytometer and aliquoted in incubation buffers at a concentration of 3 x 106 cells per 50 µl per each Eppendorf tube. To measure basal and nicotinergic 45Ca2+ influx, we used Krebs buffer (Sigma-Aldrich) supplemented to contain 1.2 mM Ca2+ ("basal" buffer; pH 7.4). Cell aliquots were resuspended in 300 µl of basal buffer containing test nicotinergic agents and 45Ca2+ (specific activity 11.6 mCi/mmol; NEN) 1% of total Ca2+, and incubated for 1 min at 37°C. After washing three times by centrifugation at 250 g for 1 min in a Beckman Coulter microcentrifuge in ice-cold, radioactive calcium-free basal buffer, the cells were solubilized in 100 µl Triton X-100 (Sigma-Aldrich), transferred into scintillation vials containing 5.0 ml of a scintillation cocktail, and 45Ca2+ taken up by the cells was measured in a liquid scintillation counter. The amount of the ligand-dependent 45Ca2+ influx was expressed as a percentage of basal influx.
AsOs assay
The phosphorothioated and FITC-tagged AsOs and the phosphorothioated, equally sized sense oligonucleotide (control) were commercially synthesized by Operon. Following the protocol provided by the manufacturer, AsOs were mixed with LipofectAMINE PLUSTM reagent (GIBCO BRL) and transfected into second passage human foreskin keratinocytes grown to 50% confluence in a standard 6-well tissue culture plate in 2.0 ml KGM. Each experimental culture received 20 nM of AsOs, and the control cultures received the same dose of control (sense) oligonucleotide, diluted in KGM containing 1.2 mM Ca2+, to induce keratinocyte differentiation (Hennings and Holbrook, 1983).
7 nAChR expression assays
To assess the effects of changes in extracellular Ca2+ concentrations on the expression of keratinocyte 7 nAChRs, keratinocytes freshly isolated from human neonatal foreskins were incubated for 0, 15, or 60 min in KGM containing 1.2 mM Ca2+ in a humid 5% CO2 incubator after which the total amount of
7 protein was measured by Western blotting (as described below), and the membrane expression of this nAChR was evaluated using FITC-labeled
-BTX (Garcia-Borron et al., 1990). Briefly, quadruplicate of experimental, i.e., 1.2 mM Ca2+-treated, and control, i.e., 0.09 mM Ca2+-treated, keratinocytes were resuspended in ice-cold PBS containing 10 µM FITC-labeled
-BTX (Molecular Probes, Inc.), incubated for 1 h at 4°C, washed three times with PBS, loaded in standard 96-well ELISA plates (Costar Corporation) at a concentration of 5 x 104/well, and the fluorescence intensity ratio (at 494 nm excitation and 518-nm emission wavelengths) was measured using the Perkin Elmer HTS 7000 instrument.
Western blot assay
Proteins were isolated from the phenol-ethanol supernatant of homogenized human or murine keratinocytes or skin samples of neonatal mice by adding 1.5 ml of isopropyl alcohol per 1 ml of Trizol Reagent (GIBCO BRL) and analyzed essentially as described in our protocol of a quantitative immunoblot assay (Arredondo et al., 2001). The specificities and working concentrations of primary antibodies used are listed in Table III. The membranes were developed using the ECL + Plus chemiluminescent detection system (Amersham Biosciences). To visualize antibody binding, the membranes were scanned with StormTM/FluorImager (Molecular Dynamics), and band intensities were determined by area integration using ImageQuant software (Molecular Dynamics). To normalize for the protein content, the housekeeping protein actin was visualized in each sample with a mouse antiactin monoclonal antibody (Sigma-Aldrich). The ratios obtained in three independent experiments were averaged to obtain the mean value (n = 3). The protein content ratio in every control (or 7 +/+) sample is always equal to 1. The images represent typical results from a series of three independent experiments.
RT-PCR assay
Total RNA was extracted from cultured keratinocytes and murine skin using guanidinium thiocyanate phenol chloroform extraction procedure (Trizol Reagent; GIBCO BRL) as described elsewhere (Chomczynski and Sacchi, 1987). 1 µg of dried, DNase-treated RNA was reverse transcribed in 20 µl of RT-PCR mix (50 mM Tris, pH 8.3, 6 mM MgCl2, 40 mM KCl, 25 mM dNTPs, 1 µg Oligo-dt [GIBCO BRL], 1 mM DTT, 1 U RNase inhibitor [Boehringer] and 10 U SuperScript II [GIBCO BRL]) at 42°C for 2 h. The PCR was performed in a final volume of 50 µl containing 1 µl of the single strand cDNA product, 10 mM Tris-HCl (pH 9.0), 5 mM KCl, 5 mM MgCl2, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, and 2.5 U Taq DNA polymerase (Perkin Elmer) and 20 pmol of each forward (5') and reverse (3') primers. To allow a quantitative determination of relative gene expression levels (Arredondo et al., 2001), the cDNA content of the samples was normalized, and the linear range of amplification was determined for each primer set. For each experiment, the housekeeping gene GAPDH was amplified with 2030 cycles to normalize the cDNA content of the samples. The amplification was performed at 94°C (1 min), 60°C (2 min), and 72°C (3 min) for 2430 cycles. The specific primers used in this study are shown in Table II. The reported ratios derived from the combination of the data obtained in three independent experiments (n = 3). The images represent typical results from a series of three independent experiments. To standardize the analysis, the gene expression ratio in the control (or 7+/+) sample is always equal to 1.
Statistics
The results of the quantitative assays were expressed as mean ± SD. Significance was determined using Student's t test.
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Footnotes |
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Acknowledgments |
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This work was supported by National Institutes of Health grants DE14173 and GM62136 and research grants from the Unilever Research-USA and Flight Attendant Medical Research Institute to S.A. Grando, and the grant SFB 547, project C2 to W. Kummer.
Submitted: 21 June 2002
Revised: 3 September 2002
Accepted: 3 September 2002
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