Abstract
TLR4 is an innate immune receptor with expression in human skin, keratinocytes as well as squamous cell carcinoma (SCC) of the skin. In the present study we investigate the role of TLR4 as a negative regulator of keratinocyte proliferation. We present here that the expression of TLR4 increased with the differentiation of cultured keratinocytes in a passage-dependent manner or under calcium-rich conditions. Moreover, the down-regulation of TLR4 by specific knockdown increased the proliferation of HaCaT keratinocytes in vitro. In addition, subcutaneously injected HaCaT keratinocytes with shTLR4 formed growing tumors in nude mice. In contrast, we observed lower proliferation and increased migration in vitro of the SCC13 cell line stably overexpressing TLR4 in comparison to SCC13 TLR4 negative cells. In vivo, SCC13 TLR4-overexpressing tumors showed delayed growth in comparison to TLR4 negative tumors. The overexpression of TLR4 in SCC13 tumor cells was followed by phosphorylation of ERK1/2 and JNK and increased expression of ATF3. In gene expression arrays, the overexpression of TLR4 in tumor cells correlated with gene expression of ATF-3, IL-6, CDH13, CXCL-1 and TFPI. In summary, TLR4 negatively regulates the proliferation of keratinocytes and its overexpression reduces tumor growth of SCC cells.
Highlights
The keratinocyte cell cycle is determined by proliferation and terminal differentiation, two processes that control and maintain the normal skin homeostasis
The total Toll like receptor 4 (TLR4) expression was analyzed by its immunoreactivity in the epidermis of normal and squamous cell carcinoma (SCC)-derived patient samples presented as tissue microarrays (TMA) and visualized by immunohistochemistry (Fig 1A–1C)
We observed a correlation in the immunoreactivity between TLR4 and IRF6 and TLR4 and Cyclin-AMP-dependent transcription factor 3 (ATF3) in moderately versus well differentiated SCC samples (Fig 1F and 1D)
Summary
The keratinocyte cell cycle is determined by proliferation and terminal differentiation, two processes that control and maintain the normal skin homeostasis. Well differentiated keratinocytes sustain the spinous, granular and corneal layers, have decreased proliferation potential and are characterized by the expression of K1, K10, involucrin, loricrin, filaggrin [1]; [2, 3]. Among the signaling pathways involved in keratinocyte differentiation are Notch [4]; Wnt [5] and p63, IRF6 [2, 6]. The dysregulation of these signaling pathways is observed in both inflammatory skin diseases such as psoriasis and non-melanoma skin cancer, such as SCC [7]; [8]; [9]; [10]
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