Expression and function of keratinocyte growth factor and activin in skin morphogenesis and cutaneous wound repair.

Reepithelialization and granulation tissue formation during cutaneous wound repair are mediated by a wide variety of growth and differentiation factors. Recent studies from our laboratory provided evidence for an important role of keratinocyte growth factor (KGF) in the repair of the injured epithelium and for a novel function of the transforming growth factor-beta superfamily member activin in granulation tissue formation. KGF is weakly expressed in human skin, but is strongly upregulated in dermal fibroblasts after skin injury. Its binding to a transmembrane receptor on keratinocytes induces proliferation and migration of these cells. Furthermore, KGF has been shown to protect epithelial cells from the toxic effects of reactive oxygen species. We have identified a series of KGF-regulated genes that are likely to play a role in these processes. In addition to KGF, activin seems to be a novel player in wound healing. Activin expression is hardly detectable in nonwounded skin, but this factor is highly expressed in redifferentiating keratinocytes of the hyperproliferative wound epithelium as well as in cells of the granulation tissue. To gain insight into the role of activin in wound repair, we generated transgenic mice that overexpress activin in basal keratinocytes of the epidermis. These mice were characterized by a hyperthickened epidermis and by dermal fibrosis. Most importantly, overexpression of activin strongly enhanced the process of granulation tissue formation, demonstrating a novel and important role of activin in cutaneous wound repair.

Tumorigenic conversion of immortal human skin keratinocytes (HaCaT) by elevated temperature.

UV-radiation is a major risk factor for non-melanoma skin cancer causing specific mutations in the p53 tumor suppressor gene and other genetic aberrations. We here propose that elevated temperature, as found in sunburn areas, may contribute to skin carcinogenesis as well. Continuous exposure of immortal human HaCaT skin keratinocytes (possessing UV-type p53 mutations) to 40 degrees C reproducibly resulted in tumorigenic conversion and tumorigenicity was stably maintained after recultivation of the tumors. Growth at 40 degrees C was correlated with the appearance of PARP, an enzyme activated by DNA strand breaks and the level corresponded to that seen after 5 Gy gamma-radiation. Concomitantly, comparative genomic hybridization (CGH) analyis demonstrated that chromosomal gains and losses were present in cells maintained at 40 degrees C while largely absent at 37 degrees C. Besides individual chromosomal aberrations, all tumor-derived cells showed gain of chromosomal material on 11q with the smallest common region being 11q13.2 to q14.1. Cyclin D1, a candidate gene of that region was overexpressed in all tumor-derived cells but cyclinD1/cdk4/cdk6 kinase activity was not increased. Thus, these data demonstrate that long-term thermal stress is a potential carcinogenic factor in this relevant skin cancer model, mediating its effect through induction of genetic instability which results in selection of tumorigenic cells characterized by gain of 11q.

Overexpression of activin A in the skin of transgenic mice reveals new activities of activin in epidermal morphogenesis, dermal fibrosis and wound repair.

Recently we demonstrated a strong induction of activin expression after skin injury, suggesting a function of this transforming growth factor-beta family member in wound repair. To test this possibility, we generated transgenic mice that overexpress the activin betaA chain in the epidermis under the control of a keratin 14 promoter. The transgenic mice were significantly smaller than control littermates, and they had smaller ears and shorter tails. In their skin, the fatty tissue was replaced by connective tissue and a severe thickening of the epidermis was found. The spinous cell layer was significantly increased, and the epidermal architecture was highly disorganized. These histological abnormalities seem to result from increased proliferation of the basal keratinocytes and abnormalities in the program of keratinocyte differentiation. After skin injury, a significant enhancement of granulation tissue formation was detected in the activin-overexpressing mice, possibly as a result of premature induction of fibronectin and tenascin-C expression. These data reveal novel activities of activin in the regulation of keratinocyte proliferation and differentiation as well as in dermal fibrosis and cutaneous wound repair.

Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization.

The development of skin carcinomas presently is believed to be correlated with mutations in the p53 tumor suppressor and ras gene as well as with the loss of chromosome 9. We now demonstrate that, in addition, loss of chromosome 15 may be a relevant genetic defect. Reintroduction of an extra copy of chromosome 15, but not chromosome 4, into the human skin carcinoma SCL-I cells, lacking one copy of each chromosome, resulted in tumor suppression after s.c. injection in mice. Transfection with thrombospondin-1 (TSP-1), mapped to 15q15, induced the same tumor suppression without affecting cell proliferation in vitro or in vivo. Halted tumors remained as small cysts encapsulated by surrounding stroma and blood vessels. These cysts were characterized by increased TSP-1 matrix deposition at the tumor/stroma border and a complete lack of tumor vascularization. Coinjection of TSP-1 antisense oligonucleotides drastically reduced TSP-1 expression and almost completely abolished matrix deposition at the tumor/stroma border. As a consequence, the tumor phenotype reverted to a well vascularized, progressively expanding, solid carcinoma indistinguishable from that induced by the untransfected SCL-I cells. Thus, these data strongly suggest TSP-1 as a potential tumor suppressor on chromosome 15. The data further propose an unexpected mechanism of TSP-1-mediated tumor suppression. Instead of interfering with angiogenesis in general, in this system TSP-1 acts as a matrix barrier at the tumor/stroma border, which, by halting tumor vascularization, prevents tumor cell invasion and, thus, tumor expansion.

Telomerase is not an epidermal stem cell marker and is downregulated by calcium.

The ribonucleoprotein complex telomerase, which was found to be active in germ line, immortal, and tumor cells, and in cells from continuously renewing normal tissues such as epidermis or bone marrow, is thought to be correlated with an indefinite life span. Therefore, it has been postulated that in the normal tissues, telomerase activity may be restricted to stem cells, the possible precursors of tumor cells. Here, we demonstrate that a 56% enriched population of epidermal stem cells exhibited less telomerase activity than the more actively proliferating transit amplifying cells, which are destined to differentiate after a finite number of cell divisions. Thus telomerase is not a stem cell marker. In human epidermis we found a heterogeneous expression of the telomerase RNA component (hTR) within the basal layer, with clusters of hTR-positive cells showing variable activities. Histone-3 expressing S-phase basal cells were distributed evenly, illustrating that hTR upregulation may not strictly be correlated with proliferation. We further show for human epidermal cells that differentiation-dependent downregulation of telomerase correlates with Ca++-induced cell differentiation and that increasing the amount of Ca++ but not Mg++ or Zn++ reduced telomerase activity in a dose-dependent manner in a cell-free system (differentiation-independent). Furthermore, addition of ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid completely reversed this Ca++-induced inhibition. These data indicate that Ca++ is not only an important regulator of epidermal differentiation but also a key regulator of telomerase.