Keratinocytes modulate the biosynthetic phenotype of dermal fibroblasts at a pretranslational level in a human skin equivalent

In this study we investigated the influence of keratinocytes on the phenotype of fibroblasts in an in vitro human skin equivalent. Keratinocytes were seeded at the surface of fibroblast-populated mechanically restrained type I collagen gels (lattices). Lattices without keratinocytes were handled in parallel as controls. After 2 and 4 days in culture, the keratinocyte layer was removed and the steady-state level of the mRNA for the main extracellular matrix macromolecules and interstitial collagenase produced by the fibroblasts was measured by Northern and dot blot analysis. A 50% decrease in the amount of procollagen type I and type III mRNAs was observed after 2 and 4 days of coculture while collagenase gene expression was upregulated by 300% when compared with control lattices. No significant modulation of type IV and type VI collagen, elastin or laminin B1 mRNA levels was found. Fibronectin mRNA levels in fibroblasts were significantly increased only on day 4. All the observed changes could be reproduced using a conditioned medium collected from a lattice covered with keratinocytes added to a lattice containing fibroblasts alone. These results indicate that in an in vitro reconstituted skin, keratinocytes are able to modulate the biosynthetic phenotype of fibroblasts at a pretranslational level through a paracrine signalling pathway.     

小鼠真皮间充质干细胞与成纤维细胞共培养对胶原分泌和TGF-β1表达的影响

目的 探讨真皮间充质干细胞在皮肤组织修复中的作用.方法 采用低血清培养基,消化-贴壁-传代法体外培养、鉴定小鼠真皮间充质干细胞(mdMSC),并与体外分离培养的正常人皮肤成纤维细胞于transwell小室培养体系中共培养,样本碱水解法和ELISA法分别检测第4、8天培养上清液中羟脯氨酸和TGF-β1的变化.结果 共培养第8天,经mdMSC 2.5×104和mdMSC 1×104处理的正常人皮肤成纤维细胞培养上清液中羟脯氨酸含量较单独培养时明显增高(P<0.05).经mdMSC处理的各组正常人皮肤成纤维细胞培养上清液中TGF-β1含量于共培养第8天时均高于单独培养(P<0.01);经mdMSC 1×104处理的正常人皮肤成纤维细胞培养上清液中TGF-β1含量在第4天亦高于单独培养,差异有统计学意义(P<0.05).各不同细胞密度的MSC处理组的羟脯氨酸含量与TGF-β1水平无相关关系(r=0.108,P＞0.05).结论 mdMSC与正常人皮肤成纤维细胞共培养可增加羟脯氨酸和TGF-β1的分泌,可能是mdMSC促进皮肤组织修复的机制之一.     

Effects of fat cells on keratinocytes and fibroblasts in a reconstructed rat skin model using collagen gel matrix culture

BACKGROUND: Fat cells (stromal tissue cells), not only have the function of lipid metabolism, but produce various cytokines that exert an influence on other cell types through paracrine or endocrine mechanisms. OBJECTIVES: To elucidate possible roles of fat cells in the skin, we examined their effects on the biological behaviour of keratinocytes and dermal fibroblasts in culture. METHODS: In the present study, focusing upon fat cell--keratinocyte or fat cell--dermal fibroblast interactions, we used a reconstructed skin system with rat skin cells in a three-dimensional collagen gel matrix culture. RESULTS: In this coculture system, fat cells promoted the proliferation and differentiation of keratinocytes. When keratinocytes were seeded directly on the fat cell layer without dermal fibroblasts, they proliferated extensively and formed a thick epidermal layer with a well-differentiated structure. Conversely, fat cells inhibited the proliferation of dermal fibroblasts. These effects of fat cells were presumed to be mediated by cytokines derived from the fat cells. CONCLUSIONS: The effects of fat cells could not be mimicked by the addition of leptin, tumour necrosis factor-alpha or insulin-like growth factor-II, suggesting that fat cells are mediating these activities via some other cytokines.     

Keratinocyte growth regulation in defined organotypic cultures through IL-1-induced keratinocyte growth factor expression in resting fibroblasts

Balanced keratinocyte proliferation and differentiation resulting in regular tissue organization strictly depend on dermal support. Organotypic cultures represent biologically relevant in vitro models to study the molecular mechanism of the underlying dermal-epidermal interactions. To mimic the state of resting fibroblasts in the dermis, postmitotic (irradiated) fibroblasts were incorporated in the collagen matrix, where they typically support epidermal proliferation and tissue organization. In coculture with keratinocytes, fibroblasts exhibit an enhanced expression of keratinocyte growth factor and the interleukin-1 receptor (type I), which further increase with culture time. In cocultured keratinocytes, keratinocyte growth factor receptor as well as RNA expression and protein release of interleukin-1alpha and interleukin-1beta are upregulated. We hypothesized that the modulated cytokine expression represents a basic mechanism for keratinocyte growth regulation. The functional significance of this double paracrine pathway, i.e., induction of keratinocyte growth factor expression in fibroblasts by keratinocytes via release of interleukin-1, was confirmed by interfering with both signaling elements: (i) interleukin-1-neutralizing antibodies and interleukin-1 receptor antagonist significantly inhibited keratinocyte growth factor release, keratinocyte proliferation, and tissue formation comparable to the effect produced by keratinocyte-growth-factor-blocking antibodies; (ii) addition of keratinocyte growth factor to cocultures with inactivated interleukin-1 pathway completely reverted growth inhibition; (iii) in organotypic cocultures with subthreshold fibroblast numbers both interleukin-1 and keratinocyte growth factor restored the impaired epidermal morphogenesis. Thus, epidermal tissue regeneration in organotypic cocultures is mainly regulated by keratinocyte-derived interleukin-1 signaling, which induces keratinocyte growth factor expression in cocultured fibroblasts. This demonstrates a novel role for interleukin-1 in skin homeostasis substantiating data from wound healing studies in vivo.     

Atopic keratinocytes induce increased neurite outgrowth in a coculture model of porcine dorsal root ganglia neurons and human skin cells

Skin of patients suffering from atopic eczema displays a higher epidermal nerve fiber density, associated with neurogenic inflammation and pruritus. Using an in vitro coculture system, allowing a spatially compartmented culture of somata from porcine dorsal root ganglion neurons and human primary skin cells, we investigated the influence of dermal fibroblasts and keratinocytes on neurite outgrowth. In comparison with dermal fibroblasts, keratinocytes induced more branched and less calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers. By adding neutralizing antibodies, we showed that nerve growth factor (NGF) and glial cell-line-derived neurotrophic factor (GDNF) are pivotal neurotrophic factors of skin cell-induced neurite outgrowth. Keratinocytes and dermal fibroblasts secreted different ratios of neurotrophic factors, influencing morphology and CGRP immunoreactivity of neurites. To investigate changes of the peripheral nervous system in the pathogenesis of atopic eczema in vitro, we analyzed neurite outgrowth mediated by atopic skin cells. Atopic keratinocytes produced elevated levels of NGF and mediated an increased outgrowth of CGRP-positive sensory fibers. Our results demonstrate the impact of dermal fibroblasts and keratinocytes on skin innervation and emphasize the role of keratinocytes as key players of hyperinnervation in atopic eczema.     

Epithelial-mesenchymal interactions enhance expression of collagen VII in vitro

Expression of collagen VII, the major structural protein of the anchoring fibrils, was assessed in vitro using indirect immunofluorescence staining and immunoblotting of collagen VII isolated from cultures. A very low level of expression was observed in monocultures of normal human fibroblasts or keratinocytes, but the expression was greatly stimulated when fibroblasts and keratinocytes were co-cultured. Primary skin explants under culture conditions supporting growth of both cell types, or mixed co-cultures of purified fibroblasts and keratinocytes, exhibited clearly enhanced synthesis of collagen VII, and the intact tissue form of this collagen could be extracted from small co-cultures. Three-dimensional skin equivalents were constructed with fibroblasts embedded in a contracted gel of collagen I and III, with an overlying stratified keratinocyte epithelium. In these equivalents, expression of collagen VII was observed primarily in the lowest epithelial cells, indicating that these cells are the main manufacturers of collagen VII. Laminin and collagen IV were deposited in a linear fashion onto the epithelial-mesenchymal interface. The results suggest that epithelial-mesenchymal interactions, either through physical interactions and/or through soluble mediators, are necessary for efficient synthesis of collagen VII and biogenesis of the anchoring fibrils.     

Keratinocytes suppress transforming growth factor-beta1 expression by fibroblasts in cultured skin substitutes

Transforming growth factor (TGF)-beta1 is a multipotent growth factor with an important role in tissue homeostasis. This growth factor regulates cell proliferation, adhesion, migration and differentiation, as well as extracellular matrix deposition. The temporal secretion and activation of latent TGF-beta1 is thus of major importance to physiological and pathological processes and in wound healing and tumour formation. Cultured skin substitutes, as used to treat extensive acute or chronic skin wounds, offer an attractive model to investigate cellular interactions in cytokine and growth factor expression and response in vitro. In the present investigation, expression of TGF-beta1 was analysed in keratinocyte, fibroblast and melanocyte monolayer cultures, as well as in the dermal vs. epidermal components of reconstituted human skin. Immunohistology, enzyme-linked immunosorbent assay (ELISA) and Northern blotting were used to demonstrate expression at the RNA and protein level. In the monolayer cultures, levels of TGF-beta1 synthesized by melanocytes were observed to be considerably elevated when compared with keratinocytes. Most TGF-beta1, however, was secreted by fibroblasts. The relative contribution of the epidermal and dermal components of the skin substitutes to overall TGF-beta1 levels was determined by comparing results obtained for either component in the presence and absence of fibroblasts and keratinocytes. From results obtained by ELISA it was apparent that TGF-beta1 levels generated predominantly by fibroblasts within the skin substitutes were greatly reduced over time in the presence of keratinocytes. Suppression of fibroblast TGF-beta1 expression in the presence of keratinocytes was also demonstrable at the RNA level by Northern blotting. Results obtained by immunohistochemistry suggest that most, if not all, of the growth factor was present in the latent form. It is therefore most likely that the observed effect results from a factor secreted by keratinocytes, which is capable of suppressing TGF-beta1 synthesis by fibroblasts. These results suggest that expression of TGF-beta1 by fibroblasts is downregulated by paracrine actions of keratinocytes in healing skin.     

Organotypic keratinocyte coculture using normal human serum: an immunomorphological study at light and electron microscopic levels

Organotypic human skin equivalents of keratinocytes and fibroblasts embedded in collagen matrix have been the subject of studies dealing with various culture conditions. Development of standardized living skin equivalents using defined culture media containing respective supplements can provide important instruments of investigation in skin biology. In addition, tissue engineering has created human skin substitutes for treatment of acute and chronic wounds. In our study, we generate a modified organotypic human skin equivalent using normal human serum instead of fetal calf serum (FCS). This living skin equivalent shows regular stratification of the epidermis and the dermal-epidermal junction zone at the light and electron microscopic level after 1 and 3 weeks of coculture. Indirect immunofluorescence reveals regular expression of differentiation antigens and the major structural proteins collagen IV, laminin 5 and the integrin chains alpha 6 and beta 4 at the dermo-epidermal junction zone. Immunoelectron microscopy demonstrates expression of collagen IV, alpha 6 and beta 4 integrin after 1 and 3 weeks of coculture. This organotypic skin model could be the basis for autologous skin grafting for acute or chronic wounds using autologous serum as well as patients' keratinocytes and fibroblasts, thus minimizing the risk of transmitting infectious agents.     

Human keratinocytes respond to interleukin-18: implication for the course of chronic inflammatory skin diseases

Interleukin (IL)-18 has been described to play a role in several inflammatory skin diseases such as eczema and psoriasis. In this study, we aimed to elucidate keratinocytes as potential targets for IL-18 effects. In human primary keratinocytes expression of IL-18Ralpha as well as responses to IL-18 were determined. In keratinocytes freshly isolated from skin biopsies of lesional atopic dermatitis or psoriasis, we observed a significantly higher expression of the IL-18Ralpha as compared with keratinocytes from normal donors. A marked upregulation was induced in vitro upon stimulation with interferon (IFN)gamma+tumor necrosis factor (TNF)alpha or poly I:C. IL-4 led to downregulation of IL-18Ralpha. IL-18-induced CXCL10/IP-10 production in freshly isolated keratinocytes from lesional psoriasis as well as in cultured normal keratinocytes. Furthermore, IL-18 upregulated major histocompatibility complex (MHC) class II expression on IFNgamma-stimulated keratinocytes. This was of functional significance as verified in coculture experiments with CD4+ T cells in the presence of superantigen. T cells produced significant amounts of IFNgamma after coculture with IL-18-induced MHC class II expressing keratinocytes. In conclusion, we have shown that keratinocytes functionally respond to IL-18 with upregulation of MHC II and production of the chemokine CXCL10/IP-10. These findings further support an important role of IL-18 in inflammatory skin diseases in the epidermal compartment.     

Epimorphin expression during human foetal hair follicle development

Epimorphin is a mesenchymal protein expressed in several organs and known to have an essential role in epithelial tissue organization, including hair follicle morphogenesis, in mice. Although about 90% homology has been reported between human and mouse epimorphin exon sequences, there is no information about expression and function of epimorphin in hair follicle development in humans. In order to elucidate the expression pattern of epimorphin in human hair follicle morphogenesis and to compare it with the distribution of tenascin and neural cell adhesion molecule (NCAM), skin samples from human foetuses of a series of estimated gestational ages (EGAs) (46-168 days EGA) were studied using monoclonal anti-epimorphin antibody MC-1, anti-tenascin antibody and anti-human NCAM antibody. Epimorphin was detected in the mesenchymal cell condensation at the pregerm stage (< 75 days EGA), and there was strong expression of epimorphin in the perifollicular mesenchymal cells around the hair germ (75-84 days EGA). At the hair peg stage (85-104 days EGA), epimorphin was around the hair peg with the strongest staining in the neck portion. This sequence of staining patterns was similar to that of tenascin. In the bulbous hair peg (105-134 days EGA), the perifollicular dermal mesenchymal cells were evenly positive for epimorphin. Mesenchymal cells underneath the follicle bulb prior to formation of the dermal papilla were also positive for epimorphin. In the lanugo hair follicle (> 134 days EGA), dermal papilla cells expressed epimorphin as well as tenascin and NCAM. These results indicate that epimorphin expression is closely linked to developing hair follicles in human foetuses. This suggests that epimorphin may have an important part in induction of morphogenesis during human foetal hair follicle development.     

Microarray analysis of gene expression in cultured skin substitutes compared with native human skin

Cultured skin substitutes (CSS), prepared using keratinocytes, fibroblasts, and biopolymers, can facilitate closure of massive burn wounds by increasing the availability of autologous tissue for grafting. But because they contain only two cell types, skin substitutes cannot replace all of the functions of native human skin. To better understand the physiological and molecular differences between CSS and native skin, we undertook a comprehensive analysis of gene expression in native skin, cultured keratinocytes, cultured fibroblasts, and skin substitutes using Affymetrix gene chip microarrays. Hierarchical tree clustering identified six major clusters of coordinately regulated genes, using a list of 1030 genes that were the most differentially expressed between groups. These clusters correspond to biomarker pools representing expression signatures for native skin, fibroblasts, keratinocytes, and cultured skin. The expression analysis revealed that entire clusters of genes were either up- or downregulated upon combination of fibroblasts and keratinocytes in cultured skin grafts. Further, several categories of genes were overexpressed in CSS compared with native skin, including genes associated with hyperproliferative skin or activated keratinocytes. The observed pattern of expression indicates that CSS in vitro, which display a well-differentiated epidermal layer, exhibit a hyperproliferative phenotype similar to wounded native skin.     

Overexpression of extracellular epimorphin leads to impaired epidermal differentiation in HaCaT keratinocytes

Epimorphin (also known as syntaxin2) is a stromal signaling factor that is temporally secreted via a non-classical route to regulate the morphogenesis of various epithelia, including skin epidermis. In this study, we show that epimorphin signaling also regulates the differentiation program in the keratinocyte. The extracellular presentation of this molecule is detectable predominantly in the dermal compartments of the skin and its secretion is increased by cell stress such as UVB irradiation, which generates an epimorphin signaling gradient in the epidermis. Artificial stimulation of functionally normal keratinocyte HaCaT cells with extracellular epimorphin triggered initiation of their differentiation program with a dramatic depression of metabolic turnover. Intriguingly, however, sustained epimorphin signaling appeared to severely attenuate the terminal cornification in the cells induced by calcium influx and anchorage-dependent anoikis. In the organotypic culture of HaCaT cells, overexpression of epimorphin impaired the successive differentiation program in the stratified epidermis-like structures; the cells underwent aberrant multicellular arrangement with a presentation of mid-differentiation markers throughout all the cell layers. These results demonstrate that inadequate epimorphin elicits an abnormal differentiation response in keratinocytes, and indicate a causal function of the epimorphin signaling gradient for the establishment of differentiated epidermal structure in the skin.     

Study of basement membrane formation in dermal-epidermal recombinants in vitro

Summary Two different dermal-epidermal recombinants were prepared in vitro and used to study the synthesis and formation of basement membrane. The first was obtained by culturing keratinocytes on the surface of a collagen lattice populated by fibroblasts. The second was prepared by coculture of both keratinocytes and fibroblasts in a collagen lattice. After 6 weeks of culture, the basal lamina was observed with electron microscopy only if keratinocytes were cultivated on top of the collagen lattice populated by fibroblasts. In the second model, however, type IV collagen, laminin, and pemphigoid bullosa antigen could be detected by immunofluorescence as well as synthesis of type IV collagen in the culture, but no basement membrane was observed by electron microscopy. These data demonstrate that, in vitro, basement membrane formation depends not only on the presence of the macromolecular components but also on the culture conditions.     

Extracellular epimorphin modulates epidermal differentiation signals mediated by epidermal growth factor receptor

BackgroundThe epidermal stratification/differentiation program is initiated in keratinocytes by a basement membrane-detachment cue and subsequently controlled by spatially and temporally regulated signaling molecules. The vital signals for the developmental behavior of the epidermis include those mediated by epidermal growth factor receptor (EGFR); however, regulatory elements responsible for activation have not yet been fully elucidated.ObjectiveThe objectives of this study were (1) to assess the effects of EGFR activation on epidermal differentiation, (2) to study the effects of epimorphin on the level of EGFR signaling degree dependent on matrix engagement, and (3) to address the impact of epimorphin modulation on EGFR-driven epidermal differentiation in a three-dimensional (3D) organotypic skin model.MethodsWe constructed skin-equivalent models with a well-stratified differentiated epidermis and utilized them to evaluate the epidermal behaviors.ResultsExtracellularly secreted epimorphin was identified as a strong candidate for signaling pathway involvement. In a 3D epidermis model, EGF stimulation was sufficient for epidermal stratification. However, overactivation of EGFR led to irregular multicellular arrangements with an abnormal differentiation profile, which appeared to be reorganized back to the normal epidermal phenotype by extracellular epimorphin. Extracellular epimorphin interestingly attenuated EGF-stimulated EGFR phosphorylation, cell growth, and migration in adherent cells. In contrast to the results of adhesion culture, extracellular epimorphin reinforced EGFR activation in suspended cells.ConclusionsThese results demonstrate that epimorphin modulates the signaling pathways mediated by EGFR for epidermal tissue organization.     

Analysis of microenvironmental factors contributing to basement membrane assembly and normalized epidermal phenotype

To understand further the role of the dynamic interplay between keratinocytes and stromal components in the regulation of the growth, differentiation, morphogenesis, and basement membrane assembly of human stratified squamous epithelium, we have generated novel, three-dimensional organotypic cultures in which skin keratinocytes were grown in the absence or presence of pre-existing basement membrane components and/or dermal fibroblasts. We found that keratinocytes cultured in the presence of pre-existing basement membrane components and dermal fibroblasts for 9 d showed rapid assembly of basement membrane, as seen by a nearly complete lamina densa, hemidesmosomes, and the polarized, linear distribution of laminin 5 and a6 integrin subunit. Basement membrane assembly was somewhat delayed in the absence of dermal fibroblasts, but did occur at discrete nucleation sites when pre-existing basement membrane components were present. No basement membrane developed in the absence of pre-existing basement membrane components, even in the presence of dermal fibroblasts. Bromodeoxyuridine incorporation studies showed that early keratinocyte growth was independent of mesenchymal support, but by 14 d, both fibroblasts and assembled basement membrane were required to sustain growth. Normalization of keratinocyte differentiation was independent of both dermal fibroblasts and structured basement membrane. These results indicated that epithelial and mesenchymal components play a coordinated role in the generation of structured basement membrane and in the regulation of normalized epithelial growth and tissue architecture in an in vitro model of human skin.     

Stromal fibroblasts from basal cell carcinoma affect phenotype of normal keratinocytes

BACKGROUND: Epithelial-mesenchymal interactions are important not only to direct the course of prenatal development of skin and its appendages but also to influence the behaviour of transformed epithelial cells. OBJECTIVES: Evaluation of the role of stromal fibroblasts on the phenotype of epithelial cells of basal cell carcinoma (BCC). METHODS: The phenotype of human BCC was compared with the in vitro model where the growth and phenotypic pattern of normal human keratinocytes were monitored in co-culture with fibroblasts prepared from stroma of BCC (BCCFs), with normal dermal fibroblasts or with two established fibroblast lines. We visualized the expression of a panel of keratins, three types of endogenous lectin [galectin (Gal)-1, Gal-3 and Gal-7], binding sites for Gal-1 and Gal-3, a proliferation marker Ki67, nucleolar protein nucleostemin (NuclS) and membrane protein Ber-EP4. A phenotype and karyotype of BCCFs were also monitored. BCCFs were also grafted to NOD/LtSz-Rag1(null) mice to evaluate their malignant potential. RESULTS: Prolonged cultivation of BCCFs has led to morphological changes, loss of contact inhibition, loss of fibroblast surface antigens and progressive aneuploidity. However, a fully malignant phenotype did not develop as these cells did not form tumours in immunodeficient mice. Co-culture of BCCFs with normal keratinocytes in vitro led to their phenotypical changes resembling those in BCC, namely, expression of keratin 19. These keratinocytes also strongly express nuclear binding sites for Gal-1 and NuclS. This phenotype was not observed when normal keratinocytes were cultured with nontumour-originated fibroblasts. CONCLUSIONS: These observations indicate that BCCFs may differ from normal fibroblasts and may play a regulatory role in BCC biology.     

Detection of melanocortin-1 receptor antigenicity on human skin cells in culture and in situ

The proopiomelanocortin (POMC) products alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH) bind to specific receptors known as the melanocortin (MC) receptors. There is increasing evidence that the MC receptor subtype 1 (MC-1R) is expressed in vitro by several other cutaneous cell types besides melanocytes and keratinocytes. Our knowledge on the MC-1R expression in skin, however, remains fragmentary. In order to examine the expression of MC-1R in human skin cells in vitro and In situ, we made use of a recently described antibody directed against the amino acids 2-18 of the human MC-1R. Flow cytometry analysis revealed the highest MC-1R antigenicity in normal melanocytes and keratinocytes, followed by dermal fibroblasts, microvascular endothelial cells and WM35 melanoma cells. Little or no expression was detected in KB carcinoma cells and Fs4 fibroblasts. In normal human skin, immunoreactivity for the anti-MC-1R antibody was detected in hair follicle epithelia, sebocytes, secretory and ductal epithelia of sweat glands, and periadnexal mesenchymal cells. Interfollicular epidermis was largely unreactive in adult skin as opposed to undifferentiated keratinocytes of fetal skin. Our findings form a framework within which MC-1 receptor expression can be studied in various skin diseases.     

Gap-junctional protein connexin 43 is expressed in dermis and epidermis of human skin: differential modulation by retinoids.

Retinoids are effective modulators of proliferation and differentiation of keratinocytes in vivo and in vitro. In mouse 10T1/2 cells, retinoid action on proliferation and neoplastic transformation is correlated with the upregulation of gap-junctional communication and expression of connexin 43 (Cx43). In the present study we have determined if retinoids induce similar effects on gene expression in human skin. Studies were conducted in intact skin and on cultured keratinocytes and dermal fibroblasts. In a clinical study, 2 weeks of treatment with 0.05% all-trans retinoic acid resulted in increased expression of Cx43 mRNA and protein in epidermis. Expression occurred predominantly in the suprabasal layer. Cultured cells exhibited a differential response to retinoic acid. In keratinocytes, increased expression of Cx43 occurred at low (10(-11) M) concentrations, whereas inhibition occurred at high (10(-7) M) concentrations; however, junctional communication, measured by dye transfer, was not altered over this concentration range. Dermal fibroblasts, in contrast, exhibited a dose-dependent increased expression of Cx43 at concentrations up to 10(-7) M retinoic acid and proportionately increased their junctional communication over this dose range. These data indicate that control of Cx43 gene expression by retinoids in human skin cells is complex. The production of gradients of junctional channels could play a role in the control of growth and differentiation in epidermis.