In vitro analyses of CD4-protein function in dedifferentiated keratinocyte cell lines
by Slavica Hristomanova Mitkovska
Date of Examination:2023-09-12
Date of issue:2023-09-06
Advisor:PD Dr. Anja Uhmann
Referee:Prof. Dr. Michael P. Schön
Referee:Prof. Dr. Ralf Dressel
Persistent Address:
http://dx.doi.org/10.53846/goediss-10088
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Abstract
English
To study the role of the Hedgehog-Patched (Hh/Ptch) signaling pathway in T-cells, the two-step DMBA/TPA carcinogenesis protocol have been applied to mice with an homozygous Ptch depletion in CD4-expressing cell (Ptch flox/flox CD4Cre+/-). This approach revealed that beside the typically DMBA/TPA-induced papilloma and squamous cell carcinoma also basal cell carcinoma developed in Ptch flox/flox CD4Cre+/- mice. These basal cell carcinoma derive from CD4+ cells of the murine skin that are characterized by their expression of the keratinocyte marker CD49f and the stem cell markers CD34 and Sca1. The progeny of these CD4+ epidermal stem cell-like cells grow permanently and increasingly with age and upon wound healing in adult mice in all epidermal layers and as multipotent hair follicle stem cells. Beside the murine skin, the existence of CD4+ epidermal cells was also confirmed in human skin in which the cells are characterized by their expression of the keratinocyte marker CD49f and the epidermal stem cell markers CD29 and K15. However, the function of the CD4 protein in epidermal stem cell-like keratinocytes is currently unknown and therefore the main aim of this dissertation was to analyze the CD4 protein function in stem cell-like keratinocytes. Thus, a dedifferentiated, keratinocyte cell line (ddHaCaT) was established, which proliferates and grows stably in low [Ca2+] levels medium, possesses a basal cell-like phenotype and differentiate upon short-term culture in high [Ca2+] level-containing medium. Unfortunately, the generation of an inducible in vitro CD4-expression system in ddHaCaT failed. Thus, the putative function of CD4 in keratinocytes was investigated by transiently overexpress CD4 in ddHaCaT cells, followed by treatment with major histocompatibility complex class II protein (MHCII), interleukin 1alpha (IL-1α), or tumor necrosis factor alpha (TNFα) and analyzing the putative role of TCR/CD3-independent CD4-mediated signaling (e.g. measurement of proliferation rate, differentiation status, intracellular cAMP levels and the cellular migration after MHCII or TNFα treatment) or CD4 expression stimuli (e.g. qRT-PCR analyses after IL-1α treatment). The results of these experiments demonstrate that (1) MHCII treatment of CD4-expressing ddHaCaT cells results in decreased proliferation and reduced cAMP levels compared to the respective controls, (2) IL-1α treatment does not impact the proliferation rate of ddHaCaT and of CD4-expressing ddHaCaT cells and, contrary to our expectations, decreased the CD4 mRNA expression of ddHaCaT cells and (3) CD4-expressing ddHaCaT actively migrate to a TNFα gradient when cultured in 2 % cFCS-containing medium. Together the data strongly hint towards a functional role of the CD4 protein in basal cell-like keratinocytes. Moreover, the data provide evidence, that CD4 functions through a TCR/CD3-independent signaling in basal cell-like keratinocytes similar as shown for monocytes and induces differentiation of basal cell-like keratinocytes. Nevertheless, future studies, with special focus on experiments using a stably transfected CD4-expressing ddHaCaT cells, are needed to confirm the finding of this work and gain further insights into the functional role of CD4 in keratinocytes. However, the cryopreserved dedifferentiated, keratinocyte cell lines can be used as basis for further studies of keratinocyte differentiation and proliferation that are crucial for the epidermal barrier maintenance, as well as disturbance of these processes that can lead to various skin diseases (e.g. psoriasis).
Keywords: CD4; Epidermal stem cells; HaCaT; Cell differentiation/dedifferentiation; Keratinocytes; IL-1α; TNFα; MHCII; cAMP
Schlagwörter: CD4; Epidermal stem cells; HaCaT; Cell differentiation/dedifferentiation; Kerationcytes; IL-1α; TNFα; MHCII; cAMP
