Translated Abstract
Background:
All-trans retinoic acid (ATRA), one of the most important derivatives of Vitamin A, has been widely used in disorders of keratinization, cosmetology, and cancer chemotherapy because of its ability to regulate gene transcription, induce cellular differentiation, proliferation and apoptosis. ATRA takes effect by various mechanisms, many are mediated by retinoic acid receptors (RARα/β/γ) and peroxidase proliferation activated receptor α/β[1]. However, during the course of clinical application, the skin redness, dryness and desquamation induced by ATRA were very common in patients, these phenomenons were accompanied by thinning and loosening of stratum corneum, and excessive trans-epidermal water loss(TEWL). And all these phenomenons were called Retinoic acid dermatitis, which can be also observed in animal model treated with ATRA[2]. Retinoic acid dermatitis is considered to be associated with abnormal epidermal barrier, whereas the specific mechanisms are still unknown[3].
Tight junctions (TJs) are the intercellular structures in epithelial and endothelial cells. In epidermal cells, TJs cooperate with adherens junctions and desmosomes to make the junctional complexes, which finally form continuously connective bands between adjacent cells [4]. In addition to effectively occluding the intercellular space between adjacent cells, TJs also play a crucial role in the regulation of diffusion of ions, solutes and the cells via the paracellular space, which enable them to regulate the epidermal permeability, inflammation and metabolism. And TJs are believed to participate in the establishment and maintenance of apico-basal polarity[5,6]. The components of TJ had just been divided and named gradually in recent years. Occludin, the first identified transmembrane component of tight junctions, has been implicated in regulating the permeability properties of tight junctions and, in particular, has been linked to the regulation of size-selective diffusion[7]. Claudin protein family is another main composition of TJs[8], studies had shown that, compared with wild type mice, claudin1-konckout mice showed very dry and red skin, companied with increased TEWL and weakened abilty of TJs to blocking tracer penetration[9]. TJs played an important role in barrier function and impaired TJs also caused epidermal barrier damage, thus, TJ molecules may be the molecular-target of all-trans retinoic acid in inducing the epidermal barrier damage during the clinic application, however there is no related literatures reported at present.
Filaggrin(Flg) is a kind of soluble acid /neutral protein in keratinocytes , and constitutes the substrate of keratin matrix. Its main function is to participate in the composition of cornified envelopes of keratinocytes in the superficial layer of epidermis, to promote terminal differentiation of granulosa cells and the formation of epidermal unique structure in stratum corneum. Moreover, Flg can be hydrolyzed very quickly to free amino acids at superficial stratum corneum layers, the latter further turns into natural moisturizing factors (NMFs), which constitute a natural moisturizing factor to matain the normal skin elasticity and permeability, and regulate the hydration of the stratum corneum[10].Atopic dermatitis and psoriasis, the two most typical diseases characterized by abnormal skin structure and function, had been confirmed to have reduced expression of Flg in skin lesions[11, 12]. Given the important role of Flg in maintaining epidermal barrier function, we speculated it may be a molecular target of all trans retinoic acid.
Objective: To study the effect of all-trans retinoic acid on epidermal tight junction molecules claudin-1, occludin and filaggrin in HaCaT cells cultured in vitro and mice skin.
Methods:
1. Quantitative real-time PCR, and western blot were used to detecte the expression of Claudin-1, occludin and Flg gene on mRNA and protein level in HaCaT cells, before and after ATRA treatment respectively.
2. Immunofluorescence staining was used to detecte the expression of claudin-1, occludin and Flg in HaCaT cells, in the absence or presence of ATRA respectively.
3. Immunofluorescence staining was used to detecte the expression of claudin-1, occludin and Flg in the skin tissue of all-trans retinoic acid dermatitis mice model and self-control.
Results:
1. Compared with blank control group, mRNA levels of claudin 1 and Flg were significantly reduced in HaCaT cells,after ATRA treatment for 24h and 36h(P ＜ 0.05), and the protein level of them were significantly reduced after ATRA treatment for 36h (P ＜ 0.05); However, mRNA level of occludin was not different between the two groups after ATRA treatment for 24h (P ＞ 0.05), but increased significantly after ATRA treatment for 36h (P ＜ 0.05), and the difference of protein level between the two groups not detected at 36h (P ＞ 0.05) but slightly increased after ATRA treatment for 48h (P ＜ 0.05)
2. The immunofluorescence staining on cells showed that Flg was localized partially in cytoplasm with a “dot” like pattern around periphery of the nucleus; claudin-1 and occludin were found positive on the membrane of cell-cell confluent conection. Compared with control group, expression of claudin-1 and Flg were decreased after ATRA treatment for 36h with statistical significance (P ＜ 0.05). While occludin expression showed no obvious difference between the two groups after 36h treatment (P ＞ 0.05), but was remarkably increased after ATRA treatment for 48h (P ＜ 0.05).
3. The immunofluorescence staining on mice skin tissue treated by ATRA showed that strongest fluorescence staining of Flg and occludin in granulosum layers. While claudin-1 stainng located on all layers except stratum corneum with weakly expressed in basal layer, but relatively strongly expressed in spinous layers and granulosum layers. Compared with the self-control, the expression of claudin-1 and Flg were both decreased in skin tissue applied with ATRA cream while the expression of occludin was remarkably increased, and the all differences were statistically significant (P ＜ 0.05).
Conclusion:
All-trans retinoic acid can significantly increase the expression of occludin and reduce expression of claudin-1 and filaggrin in HaCaT cells, which were confirmed on all-trans retinoic acid dermatitis mice model. Above results suggest that effects of all-trans retinoic acid on claudin-1, occludin and filaggrin participate in epidermal barrier damage which lead to the clinical features of retinoic acid dermatitis.
KEY WORDS: All-trans retinoic acid (ATRA); Tight juncion; Claudin-1; Occludin; Filaggrin
TYPE OF THESIS: Applied Research
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