Mechanism Of Rab23 Involved In UVB-induced Melanogenesis And Melanosome Transport

Objective: Ultraviolet(UV)-induced melanin synthesis protects the skin from UV damage,and the presence of melanin can inhibit melanoma initiation and progression,but there are also numerous studies indicating that melanin may be necessary for the malignant transformation of melanocytes,and clinicopathological analysis of advanced melanoma has shown that tumour pigmentation is inversely correlated with disease outcome(overall survival and disease-free time),The possibility that melanoma cell sensitivity to chemotherapy and radiotherapy can be improved by targeting melanogenesis was proposed as early as 50 years ago,so the study of the molecular mechanism of UV induced melanogenesis can provide new ideas for the treatment of melanoma and so on,especially to solve the problem of therapeutic resistance of tumors.Ultraviolet B(UVB)can induce melanogenesis by activating melanosome synthesis related molecules and enzymes in melanocytes,and melanosome transport is also important.Melanosomes,as a kind of membrane structural organelles,numerous studies have shown that its transport in cells is closely related to the Rab GTPase family.The Rab GTPase family is a central hub of diverse membrane trafficking processes in cells and is responsible for the biogenesis,transport,anchoring of various membrane associated organelles in cells.As a member of the Rab GTPase family,Rab23 expression levels are upregulated in UVB irradiated cells,but its relationship with UVB induced melanogenesis and melanosome transport remains unclear.Methods: B16F10 and SK-MEL-2 cell lines were selected as the research objects,and broad-band ultraviolet radiation B(BB-UVB)was used to induce melanogenesis:1)To observe the relation between Rab23 and UVB-induced melanin synthesis and transport,Western Blot was used to detect the protein level expression of Rab23 in cells after UVB irradiation,and immunofluorescence confocal was used to observe the spatial location relationship between Rab23 and melanosomes in cells after UVB irradiation;2)To determine the effect of Rab23 on UVB-induced melanin synthesis and transport,small interfering RNA(si RNA)was used to downregulate the protein expression level of Rab23 in the cells,and the experimental groups were set as si Rab23,si NC,si Rab23 + UVB irradiation,and si NC + UVB irradiation,respectively.Then the effects of si Rab23 on the melanin content and intracellular tyrosinase activity of the cells were determined by Na OH assay,tyrosinase activity assay,and DOPA staining,respectively,The dendritic length were observed and measured under inverted optical microscopy.3)To explore the effect of Rab23 affecting melanin synthesis and melanosome transport related proteins,the Western Blot was performed to detect the effects of si Rab23 on the expression of melanogenesis – related proteins like microphthalmia-associated transcription factor(MITF),tyrosinase related protein-1(TRP-1),as well as melanosome transport-related proteins Rab27 a,melanophilin(Mlph),Myosin Va;The effect of si Rab23 on melanosome transportation was observed using immunofluorescence confocal;4)Exploration of the mechanism of Rab23 involving in the regulation of UVBinduced melanin synthesis and transport: c AMP/ PKA / CREB / MITF is the key signaling pathway involved in UVB induced melanogenesis in epidermal melanocytes.To determine whether Rab23 participate in UVB-induced melanogenesis through activate the c AMP pathway,Foskolin,as a c AMP activator,was used to activate the c AMP level in the cells,and the experimental group was set as si Rab23,si NC,si Rab23 + Foskolin treatment,and si NC + Foskolin treatment.Further more,Western Blot was used to evaluate the protein level of melanogenesis-related proteins and melanosome transport-related proteins include MITF,TRP-1,Rab27 a,Mlph and Myosin Va.To further elucidate the regulatory mechanism of Rab23 on c AMP pathway,Western Blot was used to detected the phosphorylation of PKA and CREB,which is the downstream target of c AMP pathway.Results:1)UVB irradiation could induce upregulation of Rab23 protein levels in B16F10 cells;2)The Rab23 is colocalized with the mature melanosome marker TRP-1 in the cytosol of B16F10 cells after UVB irradiation;3)UVB could induce elevated melanin content,enhanced tyrosinase activity and elongated cell dendrites in B16F10 cells,while downregulating Rab23 expression in B16F10 could inhibit the effects of UVB;4)UVB could induce upregulation of protein expression levels of melanogenesisrelated molecules Microphthalmia-associated transcription factor(MITF)and Tyrosinerelated protein-1(TRP-1)as well as melanosome transport-related molecules Rab27 a,Melanophilin(Mlph)and Myosin Va in B16F10 cells and SK-MEL-2 cells,while si Rab23 inhibited the effect of UVB on melanin synthesis and melanosome transport;5)Downregulation of Rab23 expression impair in the translocation of TRP-1 to the plasma membrane,with multiple aggregates around the nucleus insdead;6)siRab23 downregulation the effect of Foskolin,which could induce the upregulation of the protein level of melanogenesis-related molecules MITF,TRP-1 and melanosome transport-related molecules Rab27 a,Mlph,myosin Va as a activator of c AMP.7)siRab23 decreases PKA and CREB phosphorylation in B16F10 cells as well as SKMEL-2 cells induced by UVB.Conclusion:UVB can induce the upregulation of Rab23 expression in cells,and the downregulation of Rab23 expression can inhibit the c AMP / PKA / CREB signaling pathway then inhibit the transcriptional activation of the MITF,which in turn downregulates the expression levels of the melanogenesis related molecule TRP-1 as well as the melanosome transport related molecules Rab27 A,MLPH,and myosin Va,inhibits the UVB induced elevation of tyrosinase activity and the transport of mature melanosomes along actin filaments,ultimately depress melanogenesis.