Serum and Extracellular Calcium Modulate Induction of Cytochrome P-45OIAl in Human Keratinocytes”

Culture conditions allowing for cytochrome P-450IA1 induction by 2,3,7,8-tetrachlorodibenzofuran (TCDF) in normal human keratinocytes (HK) were investigated. HK grown in serum-free low extracellular Ca2+ (0.1 mM) medium did not accumulate P-450IA1 mRNA in response to TCDF. If, however, the cultures were pretreated for more than 24 h with either serum or elevated extracellular Ca2+ (2.0 mM), induction of P-450IA1 was obtained by TCDF. Serum and elevated Ca2+ concentrations were found to be additive in this respect. When analyzing HK derived from five individuals, no apparent difference was found in the relative induction of P-450IA1 by increasing concentrations of TCDF, giving an EC50 of approximately 2 nM. The permissive effect of serum and elevated Ca2+ could be conferred to a reporter gene by the -1140 to +2435 part of the human CYPIA1 gene. Culture conditions allowing for P-450IA1 induction correlated with conditions that induced mRNA corresponding to the differentiation specific enzyme epidermal transglutaminase. This finding, together with the known differentiation promoting effects of serum and elevated Ca2+, suggest that terminal differentiation is necessary for P-450IA1 induction in HK by Ah receptor ligands.

Epidermal cells offer a first defense as they exhibit various detoxifying enzyme activities against potentially harmful molecules in the environment.
Polycyclic aromatic hydrocarbons (PAH)' such as 3-methylcholantrene and benz(a)anthracene have been shown to induce aryl hydrocarbon (AH) hydroxylase activity in both the epidermis and in isolated keratinocytes in vitro (l-3). Although AH hydroxylase is involved in oxidative detoxification, metabolism via this enzyme can also result in the activation of xenobiotics to toxic, mutagenic, and carcinogenic species (4). The major fraction of PAH-induced AH hydroxylase activity in rodent skin has been attributed to the CypZAl gene product, the microsomal cytochrome P-450lAl (3). *This work was supported by the Swedish National Board of Environmental Protection, the Swedish Work Environment Health Fund, and Swedish Medical Research Council Project B90-13Z-07482-05. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be herebv marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. j: To whom requests for reprints should be addressed. ' The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; AH, aryl hydrocarbon; P-450IA1, cytochrome P-450IAl; TCDF, 2,3,7,&tetrachlorodibenzofuran; HK, human keratinocytes; FCS, fetal calf serum; Me2S0, dimethylsulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAT, chloramphenicol acetyltransferase.

Polychlorinated
PAHs, such as the dioxins, dibenzofurans and biphenyls, are the most potent inducers of P-450IAl and bind the ubiquitously distributed low abundance cytosolic receptor protein (the Ah or dioxin receptor) with the highest affinity (5). The molecular mechanism of Ah receptor function has been delineated using cells of hepatic origin and is analogous to steroid receptors as it involves ligand binding, activation (including loss of the 90-kDa heat shock protein), nuclear translocation, and subsequent binding to specific DNA sequences which could lead to enhanced transcription of specific genes (6). Multiple copies of the well characterized binding motif for the liganded Ah receptor are located upstream of the rat and mouse CypZAl genes (7,8). The -1140 to +2435 region of the human CYPZAl has been shown to confer inducibility by PAHs via at least two regulatory elements to a reporter gene when transfected to a human hepatoma derived cell line (9).
The Ah receptor is a necessary, although not always sufficient, mediator of the pleiotropic biological response elicited by polychlorinated PAHs (Ref. 10 and references therein). Generally, large species variations exist in the response that include epithelial hyperplasia, thymic involution, liver damage, and teratogenic-and tumor-promoting effects. The most well established effect of polychlorinated PAHs in humans is a hyperkeratotic and metaplastic response of the hair follicles and interfollicular epidermis, leading to persistent acne-like lesions called chloracne (11).
In this study we have investigated the culture requirements for the induced expression of P-450IAl by 2,3,7&tetrachlorodibenzofuran (TCDF) in normal human keratinocytes (HK). The advantages of using HK compared with transformed cell lines are the possibility to induce differentiation and the similarity in the degree of differentiation between cell cultures established from different donors. We show that under in vitro conditions that select for an undifferentiated basal-like phenotype, TCDF cannot induce P-450IAl expression unless differentiation is allowed by raising the extracellular Ca*+ concentration and/or by treatment with serum.

MATERIALS AND METHODS
Cell Isolation and Cultiuation-All chemicals, medium, and growth factors were purchased from Sigma, unless stated otherwise. HK were isolated from adult or newborn donors as described previously (12)

RESULTS
Using culture conditions where a considerable portion of the cells showed a terminally differentiated phenotype, HK isolated from five individuals showed a comparable response with TCDF with regard to the increase in P-450IAl mRNA levels, giving ECso values of approximately 2 nM (Fig. 1, A  and B). This concentration is in agreement to that found in other systems for induction of AH hydroxylase activity (21). The time course of induction showed that maximum steadystate levels of message were achieved by 24-48 h and that the mRNA remained high throughout the 72 h of the experiment (Fig. 1, C and D).
HK can be cultivated using serum-free and low extracellular Ca*+ conditions that suppress terminal differentiation and promote rapid proliferation resulting in a homogeneous basallike population of cells. Under these culture conditions, TCDF treatment did not result in increased P-450IAl mRNA ( Fig.  2 A lanes: low calcium).
However, when differentiation was allowed by either pretreatment with elevated extracellular Ca2+ (lanes: high calcium) or serum (lanes: low calcium, serum), TCDF treatment resulted in increased P-450IAl there was an additive effect in that P-450IAl mRNA accumulated to very high levels when TCDF was present, representing at least a 30-fold induction compared with control cultures (basal levels are below the detection level). The relative induction by TCDF in serum or high Ca2+ compared with that in high Ca2+ plus serum varied to a certain degree between experiments. This is likely to be a consequence of variation in the confluency that will affect the differentiation state of HK in vitro (22) achieved to the same extent if the medium was changed back to low Ca2+ at the time of TCDF treatment.2 This effect of serum and Ca2+ did not seem to be a generalized phenomena since in the hepatoma-derived human cell line HepG2, P-450IAl mRNA was induced by TCDF regardless of culture conditions (Fig. 2B). Accumulation of P-450IAl was not detected in normal human skin fibroblasts after TCDF treatment under any of the above conditions.2 We found that a minimal Ca2+ concentration of 0.19-0.21 mM was required for P-450IAl induction (Fig. 3A) (using 5% of FCS depleted of divalent cations adds 0.02 mM or less to the Ca2+ concentration, Ref. 15). P-450IAl induction was dependent on serum concentration, and maximum induction was achieved with 5% serum (Fig. 3B). Interestingly, serum and high Ca2+ had to be present for more than 24 h before TCDF-induced levels of P-450IAl mRNA could be detected (Fig. 4).
The combined serum and high Ca2+ treatment resulted in induction of mRNA corresponding to the differentiation specific enzyme epidermal transglutaminase (Fig. 5), and this was also true, albeit to a lesser extent,, for treatments with serum and Ca2+ separately.2 Epidermal transglutaminase catalyzes the cross-linking of cornified envelope precursor proteins to form the insoluble envelope underlying the plasma membrane that characterizes the terminally differentiated keratinocyte and has been shown previously to be induced by high Ca2+ in HK (19).
In order to assess the possibility that the permissive effects of serum and high Ca2+ on inducibility of P-450IAl by TCDF could be mediated by a region involved in the transcriptional regulation of the CYPIAl gene, transient transfections were performed. HK was transfected with a plasmid construct containing CYPIAI -1140 to +2435 sequences fused to the  CAT reporter gene. Induction of CAT activity after TCDF treatment was analyzed in low Ca2+ and in cultures that had received medium containing serum, high Ca'+, or both. As shown in Fig. 6, the different pretreatments did potentiate the effect of TCDF treatment. The highest level of CAT activity was achieved when the cultures had received high Ca", serum, and TCDF which represented a 30-fold induction compared with that in low Ca'+. An enhanced CAT activity was seen in the absence of TCDF in high Ca2+ plus serum (as well as in high Ca2+ or serum) compared with the activity in low Ca*+. This effect might be related to the reported elevation of AH hydroxylase activity by high Ca*+ in murine keratinocytes in medium where serum is continually present (23). Deletion of sequences between -1140 to -49 resulted in the loss of response to TCDF and a reduction in the basal level activity (20% activity compared to the -1140 to +2435 construct in low Ca*+).

DISCUSSION
Initially we observed that TCDF treatment did not result in increased levels of P-450IAl mRNA in an undifferentiated population of normal HK and were interested to determine whether induction of differentiation would result in a responsive phenotype. An elevated extracellular Ca*+ level is generally regarded as a potent inducer of differentiation of keratinocytes in vitro (23, 24), whereas serum has been shown to inhibit the growth of HK (25). We have shown that serum or high Ca*+ pretreatment induced a state in the cells which allowed TCDF to induce P-450IAl mRNA levels and that serum and high Ca*+ together had an additive effect. The general pattern of induction of mRNA steady-state levels by TCDF using the different culture conditions could be con-ferred to a reporter gene by the -1140 to +2435 part of the gene.
The existence of a labile repressor, important in the regulation of P-450IA1, has been suggested by studies in other systems (26). Interestingly, our results are compatible with a model of derepression mediated by serum or high Ca*+ which would lead to potentiation of inducibility. In addition, genetic evidence exist for the involvement of multiple loci in the induction of P-450IAl by PAHs (27).
We favor the possibility that serum and high Ca*+ treatment results in a responsive phenotype as a consequence of a changed gene program leading to differentiation as well as an allowance of P-450IAl accumulation based on the following reasons: (i) immunohistochemically 3-methylcholantrene-induced P-450IAl has been localized to the suprabasal layers of rat skin (3) that contain cells that no longer proliferate but which are committed to terminal differentiation. This may be analogous to the herein described in uitro situation where the culture conditions allowing for P-450IAl induction also lead to an increase in mRNA levels corresponding to the differentiation specific enzyme epidermal transglutaminase. (ii) A number of differentiation markers, such as keratin Kl and KlO and filaggrin, show enhanced expression at a restricted Ca*+ concentration of 0.10-0.16 mM in mouse keratinocytes (24). We found that P-450IAl induction by TCDF occurred above a similar concentration (0.19-0.21 mM Ca*'), taking into account that HK are less sensitive to elevated extracellular Ca*+ than mouse keratinocytes (24, 28). (iii) The time required of serum and elevated Ca*+ before P-450IAl mRNA induction is permitted suggest that a sequence of cellular changes have to occur. This would agree with a time requirement for elevated Ca*+ to act before enhanced metabolism of PAHs was obtained in murine keratinocytes using culture conditions with serum constantly present (29). In HK derived from five individuals we did not detect any major variations in the EC& values of TCDF with regard to induction of P-450IAl mRNA. This system could however prove useful in studies addressing genetic variations in the human population that lead to differences in the response to Ah receptor ligands.
In addition to the species and the tissue, our results demonstrate that the cellular differentiation state is also an important modulator of the gene program altered by exposure to Ah receptor ligands. It will be important to define the limiting step/factor(s) that determine P-450IAl induction by polychlorinated PAHs in HK. Interesting future questions are how molecular mechanisms regulating P-450IAl expression correlate to changes in expression of other genes and biological effects in a cell type relevant to the human response to exposure.