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The topological relationships between erbB receptors and ligands of the epidermal growth factor family were characterized by immunocytochemistry in normal and psoriatic epidermis and in proliferating and differentiating human keratinocytes in culture. Spatial colocalization of receptors and ligands was assessed by dual immunostaining. Expression of epidermal growth factor receptor (EGFr), erbB2, and erbB3, but not erbB4, was detected throughout the epidermis, although labeling for erbB2 and erbB3 was accentuated in the upper spinous layers, and EGFr was more strongly labeled in basal cells. Of the tested growth factors, heparin-binding epidermal growth factor (HB-EGF) was diffusely expressed throughout normal and psoriatic epidermis and sparsely colocalized with EGFr in all viable epidermal layers, with increased colocalization in psoriatic epidermis. In contrast, betacellulin and heregulin/neu differentiation factor (NDF) alpha were largely restricted in their distribution to the upper spinous and granular layers. Betacellulin was downregulated in psoriatic keratinocytes. Although heregulin/NDF-beta was undetectable in normal epidermis, it was upregulated in psoriasis. Betacellulin and heregulin/NDF-alpha strikingly colocalized with EGFr and erbB3 receptors in the granular layer and in a declining gradient from the granular zone to the basal layer, respectively. Similar patterns were observed in cultured keratinocytes under proliferative conditions and upon differentiation in high-calcium medium. These morphological data collectively suggest divergent functions for members of the growth factor family, and in particular, we propose that betacellulin and heregulin/NDF-alpha are involved in epidermal morphogenesis and/or in maintenance of the differentiated phenotype.

Although originally conceived as a basis for malignant cell growth, autocrine signaling networks are currently known to be activated during tissue repair and with in vitro cultivation. In human epidermal keratinocytes, activation of the epidermal growth factor receptor by cognate ligands mediates the majority of the autonomous replicative capacity of these cells and is necessary to inhibit differentiation and apoptosis. The importance of heparin-binding growth factors in activation of this receptor was first suggested by the strong anti-proliferative effects of soluble heparin-like molecules on keratinocyte growth. This and related evidence led to the identification of amphiregulin as a major autocrine factor for keratinocytes. The binding of amphiregulin and its homolog, heparin-binding epidermal growth factor-like growth factor, to the receptor is potentially amplified by autoinduction and cross-signaling through epidermal growth factor-related polypeptides and by transmodulation of other ErbB-family receptors (HER-2, -3, -4) in cells expressing these receptors. Heparan sulfate proteoglycans and the tetraspanin family of membrane-associated proteins appear to act as cofactors in amphiregulin-driven mitogenesis mediated by the epidermal growth factor receptor, but amphiregulin's immunolocalization to keratinocyte nuclei and to filopodia may indicate other potentially novel effects. Following from the observation that amphiregulin is overexpressed in lesional psoriatic epidermis, the importance of amphiregulin in hyperproliferative skin diseases has been further supported by recent studies of the targeted expression of a transgene encoding keratin 14 promoter-driven human amphiregulin to the basal epidermis of mice. Founder transgenic mice displayed a morphologic and microscopic cutaneous phenotype that shares characteristics with psoriasis. Pharmacologic regulation of amphiregulin's expression and receptor signaling may eventually prove to be an effective strategy in the treatment of hyperproliferative skin diseases.

We investigated the regulation of mRNAs coding for acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and transforming growth factor-alpha (TGF alpha) in cultures of human neonatal foreskin fibroblasts, keratinocytes, and melanocytes. Each cell type was propagated in an optimized serum-free medium. In rapidly growing fibroblasts, the addition of fetal bovine serum caused a modest induction of aFGF message within 2 h in conjunction with a concomitant elevation of bFGF transcripts. In these same cells, TGF alpha mRNA could not be detected in any experimental condition. In contrast, keratinocytes rapidly growing in the presence of epidermal growth factor (EGF) contained transcripts for TGF alpha that increased substantially when these cells were treated with serum. This observation suggests that factors present in serum can elevate the levels of TGF alpha mRNA beyond the levels already present in keratinocyte cultures growing in the presence of EGF. These same keratinocyte cultures had low to undetectable levels of bFGF or aFGF message, and the levels of these mRNAs were not affected by serum treatment. Treatment of keratinocytes proliferating in the presence of EGF with TGF beta for 48 h caused expression of bFGF mRNA in four of six independent cell strains. TGF beta-enhanced expression of bFGF mRNA occurred as early as 12-24 h after TGF beta exposure. TGF beta did not enhance the expression of mRNA for aFGF or TGF alpha in keratinocytes. Melanocytes failed to express detectable levels of mRNA coding for any of these growth factors in the presence of absence of TGF beta or serum.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression of amphiregulin (AR) in the basal epidermis of transgenic mice [keratin 14 promoter AR gene (K14-ARGE)] has been previously shown to induce an early-onset and severe skin pathology, with many similarities to psoriasis. In this study, it is demonstrated that involucrin enhancer/promoter-dependent expression of human AR (INV-AR) in the suprabasal epidermis of transgenic mice also produces a cutaneous psoriasis-like phenotype. INV-AR mice possess a limited lifespan and scaling, papillomatous, erythematous skin with partial alopecia. INV-AR mouse histopathology also revealed epidermal hyperkeratosis, parakeratosis, acanthosis, and an exaggerated dermal vasculature. A dermal and epidermal infiltrate was also evident and consisted of both neutrophils and CD3(+) T lymphocytes. The histology of synovial joints in both the INV-AR mice and the K14-ARGE mice of our previous investigation was examined. The histologic examination revealed that 3-week-old INV-AR transgenic mice displayed normal knee joint histology, while 2- to 3-week-old K14-ARGE transgenic mice frequently displayed synovitis, as exemplified by the presence of a mixed leukocytic infiltration, increased vascularization, and enhanced deposition of fibrous matrix in the knee synovium. These results demonstrate that AR overexpression in both the basal and suprabasal epidermis of transgenic mice induces a phenotype that mimics cutaneous psoriasis, while basal AR expression is also associated with synovial inflammation, a precursor to the psoriasis-associated arthropathy, psoriatic arthritis. Collectively, the results implicate epidermal AR expression as a possible mediator of innate cutaneous immunity and epidermal proliferation and also as a potential trigger of both cutaneous psoriasis and psoriatic arthritis.

Keratoacanthoma (KA) is a variant of cutaneous squamous cell carcinoma (SCC) known for rapid growth and potential for involution. Little is known about the basis for the rapid growth because of the dearth of model systems. We hypothesized that amphiregulin (AR), a keratinocyte autocrine growth factor, had a significant role. Using immunohistochemistry, we compared 21 KA, 6 conventional SCC, and 6 basal cell carcinomas (BCC) for AR expression. All KA were positive for AR, the majority with strong immunoreactivity. The SCC were positive (5 of 6), with generally weak staining; no BCC were positive. We developed laboratory model systems to study AR overexpression in keratinocytes and its role in the pathogenesis of KA. A retroviral transduction strategy was used to overexpress AR in the HaCaT keratinocyte-like cell line. The AR overexpressing cells (HaCaT-AR) displayed autonomous proliferation in serum-free media when compared with controls (HaCaT-NIE). To develop an in vivo model, xenografts of HaCaT-AR and HaCaT-NIE were grown on SCID mice. The HaCaT-NIE cells formed thin tumors resembling conventional SCC. The HaCaT-AR cells formed rapidly growing tumors with AR expression similar to KA. HaCaT-AR cells may represent a new system for the further evaluation of KA.

Extracellular adenosine and its related nucleotides have been referred to as retaliatory metabolites that can be released into the extracellular environment during inflammation, wounding, and other pathologic states. We have previously reported that these compounds reversibly inhibit the proliferation of normal keratinocyte cultures and we now demonstrate that these compounds also arrest the proliferation of transformed keratinocytes. Although our study shows that keratinocytes express mRNA corresponding to the A2B purinoreceptors and that adenosine or AMP treatment elevates intracellular cAMP in these cells, our study also demonstrates that dipyridamole-inhibitable transport of adenosine into the keratinocyte is central to the mechanism by which adenosine and adenine nucleotides arrest proliferation in these cells. In support of this mechanism, our results demonstrate that human keratinocytes express mRNA corresponding to the recently cloned dipyridamole-sensitive human equilibrative nucleoside transporter. Interestingly, coincubation with adenosine deaminase reverses the antiproliferative action of adenosine and exerts no effect on the antiproliferative activity of the adenine nucleotides, thus supporting a model in which adenine nucleotides are enzymatically converted to adenosine and transported into the keratinocyte in a tightly coupled and adenosine-deaminase-resistant manner. Analysis of adenosine- and adenosine-monophosphate-treated keratinocytes demonstrated that quiescence is induced within 12–24 h, and fluorescence-activated cell sorter analysis suggests that treatment with these compounds may result in the inhibition of keratinocyte proliferation at both G1 and S phases of the cell cycle. In addition to their documented antiproliferative action on other cell types, adenosine, adenine nucleotides, and related analogs may also represent a potential new class of pharmacologic regulators of keratinocyte proliferation in vivo

Amphiregulin (AR) is a heparin-binding, heparin-inhibited member of the epidermal growth factor (EGF) family and an autocrine growth factor for human keratinocytes. Previ- ous studies have shown that AR expression is increased in psoriatic epidermis. To test the hypothesis that aberrant AR expression is central to the development of psoriatic lesions, we constructed a transgene (K14-ARGE) encoding a hu- man keratin 14 promoter-driven AR gene. Our results indi- cate that transgene integration and subsequent expression of AR in basal keratinocytes correlated with a psoriasis-like skin phenotype. Afflicted mice demonstrated shortened life spans, prominent scaling and erythematous skin with alope- cia, and occasional papillomatous epidermal growths. His- tologic examination revealed extensive areas of marked hy- perkeratosis with focal parakeratosis, acanthosis, dermal and epidermal lymphocytic and neutrophilic infiltration, and dilated blood vessels within the papillary dermis. Our results reveal that AR exerts activity in the skin that is dis- tinct from that of transgenic transforming growth factor- a or other cytokines, and induces skin pathology with striking similarities to psoriasis. Our observations also link the kera- tinocyte EGF receptor-ligand system to psoriatic inflam- mation, and suggest that aberrant expression of AR in the epidermis may represent a critical step in the development or propagation of psoriatic lesions. ( J. Clin. Invest. 1997. 100:2286-2294.) Key words: growth factorinflammation • skinproliferation

Previous studies have indicated that heparin differentially regulates heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR) mitogenic activity. To further explore this phenomenon, these mitogens were compared under identical cell culture conditions in two different assays. The results of our present investigation demonstrated that AR-mediated mitogenic activity in the murine AKR-2B fibroblast-like cell line was inhibited by heparin, while HB-EGF activity was enhanced. However, the absolute effect of heparin appeared to be cell type specific since HB-EGF mitogenic activity was not dramatically affected by coincubation with heparin when tested on human dermal fibroblasts. Several studies have indicated that mutation of a conserved leucine in the carboxyl-terminal region of both EGF and transforming growth factor-α results in decreased affinity for EGF receptors. Since this leucine is present in the analogous position of HB-EGF, but absent in AR, we examined the effect of deleting this residue by carboxyl-terminal truncation of HB-EGF. Analysis of recombinant forms of HB-EGF demonstrated that HB-EGF can be converted to a heparin-inhibited growth factor if the putative mature form of the protein is truncated by two residues (leucine76 and proline77) at the carboxyl terminus. Further analysis demonstrated that only leucine76 appears to be required for heparin-dependent enhancement of HB-EGF-mediated mitogenic activity, indicating that this amino acid may play a pivotal role in controlling the response of HB-EGF to heparin or related glycosaminoglycan sulfates. Our results also suggest that expression of different HB-EGF forms in vivo could result in the production of HB-EGFs with divergent responses to sulfated glycosaminoglycans and proteoglycans. © 1995 Wiley-Liss, Inc.

We previously demonstrated that human keratinocyte cultures proliferate in the absence of polypeptide growth factors (autonomous growth) and that this autonomous growth is blocked by interaction of heparin with a human keratinocyte-derived autocrine factor (KAF) which we identified as amphiregulin (AR). In the present study, we demonstrate that sulfated polysaccharides other than heparin (low and high molecular weight dextran sulfates) also inhibit the AR-mediated autonomous proliferation of human keratinocytes. Furthermore, sulfated polysaccharides such as high and low molecular weight dextran sulfates, heparan sulfate and, to a lesser extent, chondroitin sulfates B and C were also shown to be inhibitors of human keratinocyte-derived AR (k-d AR)-stimulated DNA synthesis in quiescent murine AKR-2B cell cultures. Our results demonstrate that sulfation of polysaccharides is required for AR inhibitory activity, and that several sulfated polysaccharides (other than heparin) can act as inhibitors of AR-mediated autonomous proliferation in human epidermal keratinocytes and as inhibitors of k-d AR-mediated mitogenic activity in AKR-2B cells.

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.

When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 x 10(3)/cells cm2, the cells proliferated continuously and the addition of IGF-I, EGF, TGF alpha, bFGF, or aFGF did not significantly alter growth rate. Heparin sulfate, TGF beta, or suramin inhibited keratinocyte growth factor-independent proliferation. The addition of EGF, TGF alpha, or aFGF reversed heparin-induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte-derived conditioned medium (CM) indicated the presence of TGF alpha peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte-derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti-EGF receptor mAb inhibited CM-induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte-derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR-2B cells, and EGF-receptorless mouse NR6 cells. CM-stimulated [3H]thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti-EGF receptor mAb. Heparin sulfate partially inhibited CM-induced keratinocyte clonal growth and [3H]thymidine incorporation into quiescent AKR-2B cells. We hypothesize from these data that autocrine and paracrine-acting factors produced by keratinocytes mediated their effect through the activation of both EGF receptor-dependent and EGF receptor-independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin.

Overexpression of amphiregulin (AR) has been linked to psoriasis in mouse and man. Since psoriasis is marked by hyperproliferation of keratinocytes and loss of epidermal barrier function with infiltration of inflammatory cells into the epidermis and dermis, we hypothesized that AR might contribute to the pathogenesis of psoriasis by affecting the integrity of cell-cell junctions. We find that there is a marked reduction of functional E-cadherin in psoriatic lesions from both INV-AR mice and individuals with psoriasis. Total E-cadherin levels are dramatically reduced in psoriatic lesions from INV-AR mice. Compared with normal skin, psoriatic lesions from individuals with psoriasis exhibit downregulation of the cytoskeletal-associated triton-insoluble pool of E-cadherin and the appearance of an 80 kDa ectodomain fragment in the cytoplasmic triton-soluble pool. There is reduced immunohistochemical staining for E-cadherin in the basal epidermis of human psoriatic lesions. Moreover, there is enhanced transmigration of human neutrophils through polarized epithelial cell monolayers of MDCK cells after administration of AR, but not transforming growth factor-alpha, further supporting a specific role for AR in the pathogenesis of psoriasis.

Previous investigations have shown disparate effects of adenine nucleotides on epidermal cell proliferation. Our present study demonstrates that adenosine and its related nucleotides (ATP, ADP, AMP) are antiproliferative for normal human epidermal keratinocytes cultured in the absence or presence of exogenous epidermal growth factor. Furthermore, the inhibitory effects of these compounds occur at concentrations less than 100 μM, are reversible, and do not affect the viability of the keratinocyte cultures. Our current investigation also demonstrates that both selective and nonselective adenosine receptor agonists are themselves approximately as potent as keratinocyte proliferation inhibitors, but are all less potent inhibitors than adenosine. These observations are consistent with the theory that adenosine mediates its antiproliferative response via a novel or more poorly characterized adenosine purinoreceptor subclass. Moreover, our present study demonstrates that ATP and ATP-γ-S are significantly more potent antiproliferative agents than either α,β-methylene ATP or β,γ-methylene ATP. Based on previous studies that have demonstrated that P 2y purinoreceptors possess this type of ligand specificity and that the purinoreceptor may be expressed by keratinocyte cultures, we propose that ATP may mediate its antiproliferative effects via this purinoreceptor. Collectively our results indicate that adenosine and adenine nucleotides abrogate exogenous epidermal growth factor-dependent and -independent keratinocyte proliferation at submillimolar concentrations and may be important physiologic regulators of keratinocyte growth in vivo . Further, these results suggest that these or related compounds may have application as treatments for epidermal proliferative pathologies in which the epidermal growth factor receptor-signaling pathway has been activated.