Filaggrin production by cultured human epidermal keratinocytes and its regulation by retinoic acid

Filaggrin is a basic protein normally present in the stratum corneum of epidermis. It derives from a high-molecular-weight precursor synthesized in the stratum granulosum of epidermis. This precursor, called profilaggrin, is thought to be associated with the keratohyaline granules of granular cells. It is known that profilaggrin, but not filaggrin, is present in conventional cultures of human keratinocytes grown on plastic petri dishes. In this study, we show that cultured human keratinocytes can convert profilaggrin into filaggrin, when they are grown on a collagen matrix and raised at the liquid-air interface in order to induce terminal differentiation. Moreover, the presence of terminally differentiating keratinocytes above the granular layer is necessary, but not sufficient, for the accumulation of filaggrin. Finally, we show that the accumulation of filaggrin in the outermost layers of submerged cultured human keratinocytes can be triggered by extensive removal (double delipidization) of retinoids from the serum supplement and inhibited when small concentrations (10(-11)-10(-10) M) of retinoic acid are readded to the culture medium. Altogether, the data reported suggest that not only the synthesis of profilaggrin, but also the conversion of profilaggrin into filaggrin are negatively controlled by retinoic acid. Further, it seems that retinoic acid acts directly on the conversion of profilaggrin into filaggrin rather than on the production of terminally differentiating cells capable of accumulating this protein.     

Endothelin-1 acts as an autocrine growth factor for normal human keratinocytes

Colorectal cancers are often composed of cell types representing various differentiated cell lineages, however little is known concerning the relationship of differentiation and drug resistance in these cancers. The present study was performed to develop and characterize a stable, differentiated clone of the human colon cancer cell line LS174T and to characterize the drug resistance of this cell line in relation to its undifferentiated parental cell line. LS174T cell line was treated with the differentiating agent sodium butyrate (0.5 mM) for 30 days, then recultured in standard medium. Foci of flat-appearing cells appeared and were isolated using cloning rings, and subcloned. One subclone was designated LS174T-D. The LS174T-D clone maintains a stable, differentiated phenotype in standard culture conditions in the absence of sodium butyrate. It is characterized by the formation of a polarized monolayer with dome formation and the presence of prominent apical microvilli and tight junctions. This cell line demonstrated reduced growth in soft agar and nude mice compared with the parental cell line. LS174T-D cells expressed immunoreactive intestinal mucin antigens and brush border enzymes dipeptidyl aminopeptidase (DAP)-IV and aminopeptidase. The activities of DAP-IV and aminopeptidase were increased 5.6-fold and 3.4-fold, respectively, in LS174T-D compared with parental cells. Proliferation assays demonstrated that, compared with the parental cell line, LS174T-D cells were more resistant to doxorubicin (93-fold), cisplatin (23-fold), 5-fluorouracil (12-fold), 5-fluorodeoxyuridine (31-fold), and methotrexate (12.5-fold). Intracellular uptake of (³H)-5-fluorodeoxyuridine did not differ significantly in the differentiated and undifferentiated cell lines. Levels of mdr-1 p-glycoprotein measured by Western blot and RNA Northern blot assays were also similarly low in both cell lines. However, total glutathione content and glutathione-S-transferase activities were increased in LS174T-D cells by sixfold and threefold, respectively, compared with parental cells. Depletion of glutathione by pretreatment with DL-buthionine sulfoximine reversed LS174T-D resistance to cisplatin. Long-term treatment with sodium butyrate induces or selects for colon cancer cells with features of enterocytic differentiation. This stably differentiated cell line is associated with glutathione-mediated multidrug resistance, and provides a model for further studies of differentiation in normal and cancerous colon. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

     

Vitamin D3 production by cultured human keratinocytes and fibroblasts

We have demonstrated that monolayers of human cultured newborn foreskin keratinocytes and fibroblasts elaborate vitamin D3 following exposure to UV-B. This in vitro system provides a new means to study those factors (hormones, ions, vitamin D3 metabolites, etc.) that regulate the production of vitamin D3 by human skin cells. Vitamin D3 production was enhanced greatly by using cells that were pre-treated with AY-9944, a non-toxic drug that inhibits cholesterologenesis while elevating cellular levels of 7-dehydrocholesterol, the sterol precursor of vitamin D3. The pre-D3 formed within viable, irradiated cells is transformed to D3 within a matter of hours at 37 degrees C, and keratinocytes proved to be more proficient sources of the vitamin and its metabolites than corresponding skin fibroblasts.     

AMPK regulation of the growth of cultured human keratinocytes

AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). At concentrations of 10(-4) and 10(-3) M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10(-6) M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D3 (10(-7) and 10(-6) M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p < 0.05) as keratinocytes grown in control medium went from 25% to 100% confluence. In conclusion, the data are consistent with the hypothesis that activation of AMPK acts as a signal to diminish the proliferation of cultured keratinocytes as they approach confluence. They also suggest that AMPK activators, such as AICAR and troglitazone, inhibit keratinocyte growth and that the inhibition of cell growth by 1,25-dihydroxyvitamin D3 is AMPK-independent.     

Functional vanilloid receptors in cultured normal human epidermal keratinocytes

Vanilloid receptor subtype 1, VR1, is an ion channel that serves as a polymodal detector of pain-producing chemicals such as capsaicin and protons in primary afferent neurons. Here we showed that both capsaicin and acidification produced elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured human epidermal keratinocytes. The capsaicin- and acidification-evoked increases in [Ca(2+)](i) were inhibited by capsazepine, an antagonist to VR1. VR1-like immunoreactivity was observed in the cells. These findings suggest that functional VR1-like protein is present and functions as a sensor against noxious chemical stimuli, such as capsaicin or acidification, in epidermal keratinocytes.     

Decorin gene expression and its regulation in human keratinocytes

In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.     

Production of procollagenase by cultured human keratinocytes

Using a collagen film assay utilizing 14C-labeled type I collagen, we demonstrated that cultured human keratinocytes produced a procollagenase after treatment with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Production of collagenase paralleled alterations in cellular morphology induced by TPA. When procollagenase was immunoprecipitated with antibody to human fibroblast collagenase and analyzed on sodium dodecyl sulfate-polyacrylamide gels, the zymogen was revealed as a 56- and 51-kDa doublet. The keratinocyte-derived collagenase was a neutral metalloprotease, required activation with trypsin for detection in the collagenase assay and produced the characteristic three-quarter and one-quarter length collagen cleavage products when incubated with type I collagen at 25 degrees C. The enzyme was inhibited by serum and cysteine and was largely unaffected by serine, thiol, and carboxyl protease inhibitors. Cycloheximide inhibited the TPA-induced production of collagenase, suggesting that the procollagenase was not stored preformed in the keratinocytes. Keratinocytes treated with a tumor-promoting analogue of TPA also produced collagenase, but cells treated with cytochalasin B, interleukin-1, or two non-tumor promoting phorbol esters did not. Keratinocyte-derived collagenase may play a role in wound healing and morphogenesis.     

Autocrine regulation of cell cycle progression in normal human keratinocytes

Summary Removal of competence factors insulin and pituitary extract from the culture medium, concomitant with the addition of picomolar concentrations of the late-G₁ inhibitor transforming growth factor-beta, effectively arrested cell cycle progression of normal human keratinocytes prior to their entry into the DNA synthesis phase; arrest continued for a minimum of 36 h following removal of unbound inhibitor and subsequent addition of factor-deficient medium. To demonstrate the reversibility of transforming growth factor-beta-induced arrest, two dissimilar cell populations were recruited to synthesize DNA in a predictable and reproducible manner; whereas the reinstatement of omitted competence factors induced noncycling cells to begin synthesizing DNA within 24 h, addition of keratinocyte-conditioned medium prompted an immediate progression of late-G₁ cells into S phase. Studies to determine the extent that autocrine signaling regulates cell cycle progression revealed that nontransformed keratinocytes produce an endogenous factor required for DNA replication and that production of this progression factor required competence factors insulin and pituitary extract. Keratinocyte progression factor recruited late-G₁ cells into S phase within 1–2 h, reversed transforming growth factor-beta-induced arrest in the presence of bound inhibitor, and elicited a calcium mobilization response consistent with receptor-mediated signaling. Hence, these studies demonstrate that G₁ progression of nontransformed keratinocytes into S phase requires an endogenous progression factor and suggest that this factor may direct G₁ progression by modulating the activity of a calcium-dependent kinase.     

Cell kinetic characterization of cultured human keratinocytes from normal and psoriatic individuals

Psoriasis is a chronic skin disease characterized by epidermal hyperproliferation, disturbed differentiation, and inflammation. It is still a matter of debate whether the pathogenesis of psoriasis is based on immunological mechanisms, on defective growth control mechanisms, or possibly on a combination of both. Several in vivo cell biological differences between psoriatic lesional epidermis and normal epidermis have been reported. However, it is not clear whether these changes are causal or consequential. In case that keratinocytes from psoriatic patients have genetically determined deficiencies or polymorphisms with respect to autocrine growth regulation and the response to inflammatory cytokines, we hypothesize that these differences should be maintained in culture. Here we have started a systematic comparison of first passage keratinocytes cultured from normal skin and uninvolved psoriatic skin to address the question whether there are intrinsic differences in basic cell cycle parameters. In an established, defined culture system using keratinocyte growth medium (KGM) we have determined: (i) cell cycle parameters of exponentially growing keratinocytes, (ii) induction of quiescence by transforming growth factor β₁ (TGF-β₁), and (iii) restimulation from the G₀-phase of the cell cycle. Bivariate analysis of Iodo-deoxyuridine incorporation and relative DNA content was performed by flow cytometry. Within the limitations of this model no gross differences were found between normal and psoriatic keratinocytes with respect to S-phase duration (T_s), total cell cycle duration (T_c), responsiveness to TGF-β₁ and the kinetics for recruitment from G₀. In psoriatic keratinocytes we found a lower amount of cells in S-phase and a shorter duration of G₁, compared to normal keratinocytes. The methodology developed here provides us with a model for further studies on differences between normal and psoriatic keratinocytes in their response to immunological and inflammatory mediators. © 1996 Wiley-Liss, Inc.     

Autocrine induction of substance P mRNA and peptide in cultured normal human keratinocytes.

In this study, we have demonstrated that normal cultured keratinocytes (KCs) could generate significant endogenous substance P (SP) in a dose- and time-dependent response to exogenous SP by sensitive ELISA assay and express preprotachinin-a mRNA by RT-PCR and Southern blotting. We performed immunohistochemical analysis to confirm the presence of SP in cultured keratinocytes. In contrast, adrenaline, acetylcholine, histamine and CGRP induced only low amount of SP from cultured normal human KCs. This is the first report that SP can be induced by skin epithelial cells in response to exogenous SP and KC derived SP might play an important role in induction and acceleration of certain cutaneous diseases.     

Differentiation of normal and tumoral human keratinocytes cultured on dermis: Reconstruction of either normal or tumoral architecture

Summary Normal human keratinocytes isolated from skin and squamous carcinoma cells established from a human tumor (TR146 cell line) both exhibit limited morphologic differentiation when they are grown on conventional plastic dishes. However, when they are seeded on human de-epidermized dermis and cultured at the air-liquid interface, they are able to reform an epithelium having the morphology of the tissue of origin (i.e. skin or squamous carcinoma). The distribution in such reconstructed tissues of differentiation markers such as bullous pemphigoid antigen, 67K keratin, involucrin, membrane-bound transglutaminase, and filaggrin was very similar to their distribution in normal skin and squamous carcinoma specimens, respectively. The degree of differentiation is for both cell types extremely sensitive to culture conditions such as retinoic acid concentration, emersion of the cultures, etc. These results show that subcultured normal or tumoral keratinocytes are able to recover their specific morphogenetic potential when cultured in an environment close to their in vivo situation.     

The radiosensitivity of cultured human and mouse keratinocytes

Clonogenic survival assays after gamma-radiation in vitro were performed on freshly isolated and subcultured keratinocytes from mouse skin, mouse tongue and human skin. Survival curves were constructed by fitting the data to a multi-target model of cell survival. When subcultured, keratinocytes from all sites produced survival curves which showed a reduced shoulder region and an increased D0 when compared with their freshly isolated counterparts. Freshly isolated human skin keratinocytes were more radiosensitive than mouse keratinocytes from either skin or tongue.     

Serine-palmitoyl transferase activity in cultured human keratinocytes

Sphingolipids comprise approximately 25% of the stratum corneum lipids and are considered critical constituents of the epidermal permeability barrier. Whether sphingoid base structures are synthesized in the epidermis or whether they are derived from circulating or dermal sources is not known. We report here the initial characterization of serine-palmitoyl transferase (EC 2.3.1.50; SPT), the rate-limiting enzyme in the synthesis of sphingolipids, from cultured human neonatal keratinocytes. Subcellular fractionation studies demonstrated that 79% of the total cellular SPT activity was associated with the microsomes. The specific activity of keratinocyte SPT was 270 +/- 20 pmol/min per mg of microsomal protein, a level significantly higher than activities reported in other tissues. Keratinocyte SPT showed an apparent Km for L-serine of 0.40 (+/- 0.04 mM, with an alkaline pH optimum (8.2 +/- 0.4). Keratinocyte SPT utilizes palmitoyl-CoA preferentially over other saturated or unsaturated acyl-CoA substrates; increasing acyl-CoA chain lengths above C16 by one or two carbons was less detrimental to activity than similar decrements in chain length. Finally, the mechanism-based inhibitors L-cycloserine and beta-chloro-L-alanine, demonstrated potent inhibition of keratinocyte SPT activity, with 50% inhibitory concentrations of approximately 3.0 and 25 microM, respectively. In summary, we have found that cultured human neonatal keratinocytes contain unusually high levels of serine-palmitoyl transferase activity, and that the substrate specificity of keratinocyte SPT may determine the base composition of epidermal sphingolipids.     

Homocysteine decreases endothelin-1 production by cultured human endothelial cells.

Hyperhomocysteinemia is believed to be responsible for the development of vascular disease via several mechanisms, including the impairment of endothelial-cell functionality. In-vitro studies have demonstrated that homocysteine decreases the production or bioavailability of vasodilator autacoids, such as prostacyclin and NO. Here, we show that the treatment of human endothelial cells with noncytotoxic homocysteine concentrations leads to a dose-dependent decrease in both the secretion of the vasoconstrictor agent endothelin-1 (ET-1) and the level of its mRNA. Homocysteine had an inhibitory effect at pathophysiological (0.1 and 0.5 mmol.L(-1)) and pharmacological noncytotoxic (1.0 and 2.0 mmol.L(-1)) concentrations. Mean percentage variation from control for ET-1 production was -36. 2 +/- 18.9% for 0.5 mmol.L(-1) homocysteine and -41.5 +/- 26.8% for 1.0 mmol.L(-1) homocysteine, after incubation for 8 h. Mean percentage variation from control for steady-state mRNA was -17.3 +/- 7.1% for 0.5 mmol.L(-1) homocysteine and -46.0 +/- 10.1 for 1.0 mmol.L(-1) homocysteine, after an incubation time of 2 h. ET-1 production was also reduced by incubation with various other thiol compounds containing free thiol groups, but not by incubation with thiol compounds with no free thiol group. Co-incubation of cells with homocysteine and the sulfhydryl inhibitor N-ethylmaleimide prevented the effect of homocysteine on ET-1 production, confirming a sulfhydryl-dependent mechanism. Based on the reciprocal feedback mechanism controlling the synthesis of vasoactive mediators, these preliminary data suggest a mechanism by which homocysteine may selectively impair endothelium-dependent vasodilation by primary inhibition of ET-1 production.     

Growth-inhibitory effects of 1,25-dihydroxyvitamin D3 on normal human keratinocytes cultured in serum-free medium

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the growth of normal human keratinocytes cultured in serum-free medium was investigated. 1,25(OH)2D3 inhibited the cell growth at 10(-7) M by 75.3% and at 10(-6) M almost completely. The growth inhibition was accompanied by changes related to proliferation: (1) remarkable inhibition of DNA synthesis, (2) the decrease in the number of high-affinity receptors for epidermal growth factor, with almost no change in total receptor number, (3) the rapid decrease in c-myc mRNA level. The inhibition of DNA synthesis and the decrease of c-myc mRNA expression occurred at 3 h after the addition of 1,25(OH)2D3. These results suggest that decrease of c-myc mRNA expression is one of the primary effects of 1,25(OH)2D3 in the growth inhibition of human keratinocytes.     

Expression of cathepsin B-like enzyme activity in cell lysate from cultured human normal keratinocytes.

In the present study, we have found that the cell lysate from cultured human normal keratinocytes from foreskin (HFKs) hydrolyzed alpha-N-benzoyl-DL-arginine beta-naphthylamide (BANA), and the BANA hydrolysis occurred most under conditions of 37 degrees C and pH 6.0. This activity was strongly inhibited by leupeptin, which is an inhibitor to cathepsin B. These results suggested that the cell lysate from cultured HFKs contained cathepsin B-like enzyme activity. This is the first report to demonstrate that cathepsin B-like enzyme activity was expressed in the cell lysate from human normal keratinocytes.