The Influence Of Sonic Hedgehog Signaling Pathway On The Proliferation And Apoptosis Of Human Keratinocyte And Its Molecular Mechanisms

Keratinocyte(KC) is the main cutaneous component cell(>80%) which plays a key role in cutaneous structure and various physiological and pathologic processes of skin.The excessive proliferation and insufficient apoptosis of KC lead to a variety of chronic inflammatory skin diseases and malignant skin cancers. Therefore, the regulatory mechanisms of KC proliferation and apoptosis are always the academic focus in dermatologic researches.More and more data suggest that the classical signaling pathways involved in the embryo development control the cellular growth, proliferation, differentiation and apoptosis, such as sonic hedgehog(Shh) signaling pathway. Abundant data and our work indicate that Shh pathway controls the proliferation of normal KC, and the inhibition of Shh pathway suppresses the excessive proliferation and enhances apoptosis of KC.However, the involved regulatory mechanism is still unclear.We know the cellular metabolism is not modulated by several sporadic molecules simply, but is under the control of many orderly, complex and coherent signaling networks.Therefore, in order to clarify the regulatory mechanism of Shh pathway in KC proliferation and apoptosis, the most primary work is to identify the pathway via which Shh pathway plays its part in KC metabolism. Many recent data indicate that there exists close links between Shh signaling and extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase(MEK/ERK or ERK) pathway signaling cascade which controls cell proliferation, differentiation or death and is one of the most studied intracellular pathways. Thus we hypothesized that MEK/ERK pathway might be important in the modulation of Shh signaling in KC metabolism. Besides, Shh signaling can induce the epidermal growth factor(EGF) expression in Ha Ca T keratinocyte;meanwhile, EGF can modulate the expression of Gli-1(transcription factor of Shh pathway) in KC in return. That is to say, there exists intimate interaction between Shh and EGFR pathways, too; moreover, EGFR signaling pathway is the direct upper stream of MEK/ERK cascade, which further reminds us that MEK/ERK signaling cascade may play a critical part in the modulation of Shh signaling in the biological characteristics of KC.Similar report has not been seen yet.Our object was just to elucidate the following relationships: 1. the influence of Shh signaling pathway on normal KC proliferation and apoptosis; 2. the regulatory mechanism of Shh signaling pathway in KC proliferation and apoptosis; 3. furthermore, to investigate the relations existing between Shh and EGFR signaling pathways.Methods:Normal human primary epidermal KCs(NHEKs) and human Ha Ca T keratinocytes were cultured in vitro, respectively. RT-PCR and western blot were applied to detect the m RNA/protein levels of the key molecules of Shh pathway(Ptch-1 and Gli-1) in KC.CCK-8 assay was applied to evaluate cell growth. Flow cytometry was used to analyze the cell cycle distribution and apoptosis rates, after cells were labelled with propidium iodide(PI) or doubly labelled with Annexin V-FITC, respectively. Real time PCR and western blot were applied to detect the m RNA/protein levels of the molecules observed,respectively: we performed the following observations in turn: the influence of the modulation of Shh pathway on the expression of p-ERK1/2, p-AKT, p-STAT1 andp-STAT3 in KC; and the influence of the block of MEK/ERK activation on Shh signaling-induced expression of cyclin D1 and Bcl2; and the influence of the combined modulation of Shh/EGFR pathways on the Shh signaling-induced expression of p-ERK1/2, cyclin D1 and Bcl2 in KC.Results:1. Shh signaling pathway is activated in normal KCs. Rh Shh enhanced the proliferation and inhibited apoptosis by the upmodulation of Shh pathway activity, and cyclopamine restrained proliferation and induced apoptosis by the inhibition of Shh pathway activity in normal KCs. The upregulation and downregulation of Shh pathway activity induced and suppressed the m RNA/protein levels of cyclin D1 and Bcl2,respectively.2. Shh pathway activated MEK/ERK cascade but did not affect the PI3K/AKT,JAK/STAT1 and JAK/STAT3 pathways in normal KCs. The block of MEK/ERK activation by U0126 abolished the modulation of Shh signaling pathway on KC proliferation and apoptosis, and abolished Shh signal-induced ERK phosphorylation and the expression of cyclin D1 and Bcl2 at m RNA/protein levels in normal KCs.3. EFGR signaling modulated the Shh pathway activity in normal KCs in vitro by affecting the stability of Gli-1 and Gli-2 as well as the level of Ptch-1, the receptor and target gene of Shh pathway. Combined modulation of Shh and EGFR signaling pathways acted additively to induce the ERK activation and the increases in cyclin D1 and Bcl2 in normal KCs in vitro.Conclusions:1. Shh pathway-mediated cyclin D1 and Bcl2 alterations are involved in the proliferation and apoptosis of KCs in vitro.2. The Shh signaling pathway specifically activates MEK/ERK signaling, but does not affect the PI3K/AKT or JAK/STAT pathways in KCs in vitro.3. Shh signaling induces proliferation and inhibits apoptosis in normal KCs via cyclin D1 and Bcl2 in a MEK/ERK dependent manner.4. Shh acts additively with EGFR signaling to induce ERK activation and theexpression of cyclin D1 and Bcl2, and is stimulated by EGFR signaling in KCs in vitro.To conclude, this previously unclarified cross-talk between Shh and MEK/ERK pathways is a required channel by which Shh signaling pathway produces its modulation in normal KC proliferation and apoptosis. Which is significant for further clarifying the regulatory mechanism of Shh pathway in normal KC metabolism and helpful for the ultimate demonstration to the pathogenesis of many skin diseases.