Nicole Maas-Szabowski

c-Jun and JunB Antagonistically Control Cytokine-Regulated Mesenchymal–Epidermal Interaction in Skin

Interactions between mesenchymal and epithelial cells are responsible for organogenesis and tissue homeostasis. This mutual cross-talk involves cell surface proteins and soluble factors, which are mostly the result of regulated transcription. To elucidate dimer-specific functions of the AP-1 family of transcription factors, we reconstituted skin by combining primary human keratinocytes and mouse wild-type, c-jun(-/-), and junB(-/-) fibroblasts. We have discovered an antagonistic function of these AP-1 subunits in the fibroblast-mediated paracrine control of keratinocyte proliferation and differentiation, and traced this effect to the IL-1-dependent regulation of KGF and GM-CSF. These data suggest that the relative activation state of these AP-1 subunits in a non-cell-autonomous, transregulatory fashion directs regeneration of the epidermis and maintenance of tissue homeostasis in skin.

The lysosomal cysteine protease cathepsin L regulates keratinocyte proliferation by control of growth factor recycling

Mice deficient for cathepsin L (CTSL) show epidermal hyperplasia due to a hyperproliferation of basal keratinocytes. Here we show that the critical function of CTSL in the skin is keratinocyte specific. This is revealed by transgenic re-expression of CTSL in the keratinocytes of ctsl-/- mice, resulting in a rescue of the ctsl-/- skin phenotype. Cultivation of primary mouse keratinocytes with fibroblast- and keratinocyte-conditioned media, as well as heterologous organotypic co-cultures of mouse fibroblasts and human keratinocytes, showed that the altered keratinocyte proliferation is caused primarily by CTSL-deficiency in keratinocytes. In the absence of EGF, wild type and CTSL-knockout keratinocytes proliferate with the same rates, while in presence of EGF, ctsl-/- keratinocytes showed enhanced proliferation compared with controls. Internalization and degradation of radioactively labeled EGF was identical in both ctsl-/- and ctsl+/+ keratinocytes. However, ctsl-/- keratinocytes recycled more EGF to the cell surface, where it is bound to the EGF-receptor, which is also more abundant in ctsl-/- cells. We conclude that the hyperproliferation of keratinocytes in CTSL-knockout mice is caused by an enhanced recycling of growth factors and growth factor receptors from the endosomes to the keratinocyte plasma membrane, which result in sustained growth stimulation.

Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-α

The human keratinocyte cell line HaCaT expresses essentially all epidermal differentiation markers but exhibits deficiencies in tissue organization as surface transplants in nude mice and even more so in organotypic co-cultures with fibroblasts. Whereas tissue differentiation by normal keratinocytes (NEKs) is regulated by stromal interactions, this mechanism is impaired in HaCaT cells. This regulatory process is initiated by interleukin-1 (IL-1) release in keratinocytes, which induces expression of keratinocyte growth factor (KGF/FGF-7) and granulocyte macrophage-colony stimulating factor (GM-CSF) in fibroblasts. Production and release of IL-1 is very low and, consequently, expression of the fibroblast-derived growth factors KGF/FGF-7 and GM-CSF is absent in HaCaT-fibroblast co-cultures. However, addition of KGF and GMCSF, respectively, is inefficient to improve stratification and differentiation by HaCaT cells due to the low expression of their cognate receptors. More importantly, expression and release of the autocrine keratinocyte growth factor TGF-α is dramatically decreased in HaCaT cells. Addition of TGF- α or EGF stimulated HaCaT cell proliferation but, even more effectively, suppressed apoptosis, thus facilitating the formation of a regularly stratified epithelium. Furthermore, TGF-α enhanced the expression of the receptors for KGF and GM-CSF so that addition of these growth factors, or of their inducer IL-1, further improved epidermal tissue differentiation leading to in vitro skin equivalents comparable with cultures of NEKs. Thus, supplementing TGF-α normalized epidermal tissue regeneration by immortal HaCaT keratinocytes and their interaction with stromal cells so that regular skin equivalents are produced as standardized in vitro models.

Increased keratinocyte proliferation by JUN-dependent expression of PTN and SDF-1 in fibroblasts

In skin, fibroblasts of the connective tissue play a decisive role in epidermal homeostasis and repair by contributing to the regulation of keratinocyte proliferation and differentiation. The AP-1 transcription factor subunit JUN plays a crucial role in this mesenchymal-epithelial interplay by regulating the expression of two critical paracrine-acting cytokines, keratinocyte growth factor (KGF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We have performed gene expression profiling of wild-type and Jun–/– mouse embryonic fibroblasts to identify additional players involved in this complex network, and have found pleiotrophin (PTN) and the stromal cell-derived factor 1 (SDF-1) as novel JUN-regulated factors. Both cytokines are expressed by dermal fibroblasts in vivo, as shown by semi-quantitative RT-PCR and in situ hybridization on murine skin sections. Using a heterologous feeder layer co-culture system, we demonstrated that PTN and SDF-1 exert a mitogenic effect on primary human keratinocytes. Moreover, SDF-1-induced keratinocyte proliferation could be specifically inhibited by neutralizing antibodies against SDF-1 or its receptor, CXCR4. Consistent with its role in promoting keratinocyte growth, PTN was upregulated during cutaneous wound healing in vivo. Interestingly, co-cultivation with keratinocytes stimulated PTN expression but repressed SDF-1 production in fibroblasts, demonstrating the complexity of the paracrine regulatory cytokine networks that control skin homeostasis and regeneration.

Targeting perlecan in human keratinocytes reveals novel roles for perlecan in epidermal formation

Heparin-binding growth factors are crucial for the formation of human epidermis, but little is known about the role of heparan sulfate proteoglycans in this process. Here we investigated the role of the heparan sulfate proteoglycan, perlecan, in the formation of human epidermis, by utilizing in vitro engineered human skin. By disrupting perlecan expression either in the dermis or the epidermis, we found that epidermally derived perlecan is essential for epidermal formation. Perlecan-deficient keratinocytes formed a strikingly thin and poorly organized epidermis because of premature apoptosis and failure to complete their stratification program. Exogenous perlecan fully restored epidermal formation. Perlecan deposition in the basement membrane zone correlated with formation of multilayered epidermis. Perlecan deficiency, however, had no effect on the lining and deposition of major basement membrane components as was evident by a continuous linear staining of laminin and collagen IV. Similarly, perlecan deficiency did not affect the distribution of β1 integrin. Addition of the perlecan ligand, fibroblast growth factor 7, protected perlecan-deficient keratinocytes from cell death and improved the thickness of the epidermis. Taken together, our results revealed novel roles for perlecan in epidermal formation. Perlecan regulates both the survival and terminal differentiation steps of keratinocytes. Our results suggested a model whereby perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors involved in epidermal morphogenesis.

Organotypic cocultures as skin equivalents: A complex and sophisticated in vitro system

To assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, we have established in vitro skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination of cells derived from mouse and human tissues facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and thus the precise analysis of growth regulatory mechanisms.

Experimental Models to Analyze Differentiation Functions of Cultured Keratinocytes In Vitro and In Vivo

In this chapter, we present technical details for the generation of in vitro skin equivalents consisting of collagen gels with incorporated fibroblasts covered by proliferating and differentiating keratinocytes. Epithelial-mesenchymal interactions are clearly manifest in these skin equivalents. Therefore, they have proven to be suitable experimental tools for a broad range of applications, e.g., for studies on the the paracrine regulation of keratinocyte differentiation and proliferation. On the other hand, in vivo assays cannot be abandoned totally, in particular, when such properties as malignant growth potential, disturbed differentiation control in carcinogenesis, and impact on angiogenesis are concerned. For that reason, we additionally describe xenotransplantation techniques to graft human keratinocytes and skin equivalents, respectively, onto the dorsal muscle fascia of thymus-aplastic mice.

Alpha 9 acetylcholine receptors are essential for epidermal differentiation

Extraneuronally, acetylcholine (Ach) is synthesized, stored and secreted in the tegumental cells covering the inner and outer surfaces of the body. It acts via nicotinic (nAchR) and muscarinic (mAchR) receptors in a paracrine and autocrine fashion influencing various keratinocyte functions. Using in vitro studies, we present evidence that effects of cholinergic agonists and antagonists on cutaneous biology are dependent upon specific Ach‐R, localised in the different compartments of the skin. Using immunohistochemistry on frozen section of normal skin we could previously demonstrate a differential distribution of alpha 3*, alpha 7, alpha 9 and beta 1 nAchR and m1, m3–5 mAchR in human epidermis. Here we show the functional significance of AchR blockage using organotypical keratinocytes cultures. Cholinergic agonists and antagonists were added on the day of the lift to the air‐liquid interface. After 5 days, blockage of all AchR using mecamylamine and atropine resulted in complete inhibition of terminal differentiation. Mecamylamine and atropine alone had only a retarding effect suggesting an additive mechanism of nAchR and mAchR blockage. Specific inhibition of α9 homooligomers with strychnine produced the strongest effects leaving behind only a keratinocytes monolayer. Stimulation of AchR with either nicotine or muscarine in short term organotypic cultures did not produce dramatic changes of epidermal morphology. We conclude that alpha 9 AchR that are located in the basal and lower suprabasal layers of the epidermis, are crucially involved in terminal differentiation of keratinocytes. The functional significance of other nAchR expressed in the epidermis remains unclear, due to the lack of specific agonists and antagonists.