| BackgroundActin related proteins (Arps) constitute a novel, evolutionarily conserved superfamily of proteins whose primary sequence displays significant similarity to the conventional actins, which are enigmatic components of many chromatin-remodeling enzyme complexes with conventional actins. In addition to their role as components of cytoskeleton, recent studies revealed that Arps can actually impact transcription. Nuclear Actin-Like6a (ACTL6a, also known as Arp4or BAF53) can bind core histones, and facilitate the interaction of the chromatin remodeling complexes with nucleosomes, which plays crucial roles in controlling gene expression, DNA replication and other chromatin transactions. In the epidermis, a tissue that balances stem cell self-renewal with differentiation, stem cells in the innermost epidermal basal layer constantly differentiate and form the spinous layer, in which cells express the key cytoskeletal proteins keratin1(K1) and10(K10). As differentiation progresses, the cells generate the granular layer, which is characterized by the expression of fillaggrin and loricrin. Rising studies have focused on the role of ACTL6a as key regulators of stem cell functions. Recent discovery suggests that conditional loss of ACTL6a results in epidermal terminal differentiation, cell cycle exit and hypolasia while enforced ACTL6a expression promotes the progenitor state and suppresses differentiation. Mammalian ACTL6a and β-actin, as components of the SWI/SNF-like BAF chromatin-remodeling complex, alter nucleosome architecture, either by covalent modification of the N-terminal histone tails or by ATP-dependent perturbations of histone-DNA interactions. ACTL6a maintains the undifferentiated progenitor state by opposing SWI/SNF-enabled activation of KLF4and other epidermal differentiation genes. However, molecular functions of nuclear ACTL6a remain largely uncharacterized in epidermal keratinocytes.Histone modifying enzymes, chromatin-remodelling complexes, histone methylation and acetylation are thought to be components of intricate epigenetic mechanisms that help compact and organize genomes into discrete chromatin domains. Especially, lysine methylation coordinates the recruitment of chromatin modifying enzymes and plays an important role in the dynamics of chromatin organization during cell differentiation, apoptosis and organ development. To date, genome-wide DNA methylation has been identified as the main epigenetic change leading to the onset and progression of various diseases. Psoriasis is considered to be a multifactorial epidermal abnormalities-related disease with distinct hyperproliferation of the normally quiescent basal keratinocyte population which contains the keratinocyte stem cells. Increasing abnormal promoter methylation in cancer suppressor genes and immune-related genes has been found in psoriatic lesions. However, very little is known about how the methylation patterns and cellular activities are precisely controlled by ACTL6a in psoriasis. Here we examined the expression ACTL6a in normal and psoriatic skin, its role in epigenetic regulation role and cellular activities to unveil its potential role in psoriasis pathogenesis. [Objective]Our aim in this paper is to confirm the expression of ACTL6a on normal skin and psoriatic skin and the KCs from the respective tissue and investigate whether ACTL6a acts in the chromatin reassembly cooperative with HP1γ; we’ll investigate whether ACTL6a elevation in KCs involves in nuclear epigenetic regulation and expression of a series of keratins; furthermore, we will clarify the effects of elevated ACTL6a on the cellular proliferation, migration, senescence and ROS of epidermal keratinocytes.[Methods]1. Detect and compare the distribution of ACTL6a and HP1γ in normal skin, psoriatic lesional and perilesional skin and non-lesional skin by use of tissue immune fluorescent technology. Then we detect the distribution and expression of KCs from the respective tissues by use of cellular immunofluorescent technology and Western blot.2. Lentivirus-induced ACTL6a elevation assays were conducted. Western-blot and RT-PCR were used to determine the mRNA and protein expression of the lentivirus-treated KCs and the control lentivirus-treated KCs. Then we detected the localization and expression of HP1γ in ACTL6a-elevated KCs compared with the control lentivirus-treated KCs.3. We detected the localization of methylated histones and their global level changes in ACTL6a-elevated KCs compared with the control lentivirus-treated KCs. We also investigated its global acetylation state after ACTL6a elevation by western blot.4. Its regulation of ACTL6a elevation in keratin1,2,10,14,16, involucrin and loricrin was examined. MTS assays were used to determine the effect of ACTL6a elevation on the proliferation. Transwell assays were used to determine the effect of ACTL6a elevation on the migration of normal KCs. Cellular senescence and ROS assays were conducted to examine the role of elevated ACTL6a on senescence and ROS. [Results]1. Expression and localization of ACTL6a and HP1γ in normal skin, psoriasis skin, and KCs from these tissues.(1) Tissue immunofluorescence:ACTL6a and HP1γ expressed in all epidermal layers in normal and psoriatic skin; ACTL6a expressed within cell nuclei in normal and psoriatic skin, but HP1γ showed different staining pattern in normal and psoriatic skin.(2) Western blot:Cultural KCs from psoriatic peri-lesional skin expressed higher levels of ACTL6a and HP1γ protein than KCs from psoriatic lesional skin, non-lesional skin and normal skin.(3) RT-PCR:The mRNA expression of ACTL6a in cultural KCs from psoriatic lesional skin was higher than that from normal skin.2. Overexpression of ACTL6a prompted tether of HPly to the periphery of nucleiIn normal epidermal keratinocytes, HPly showed mainly a homogeneous nuclear staining. As compared with a mainly pericentric staining patterns of HP1γ in control LV, Actl6a LV tethered HP1γ in the periphery-nuclear regions and the perinucleolar regions. However, the protein expression of HPly in ACTL6a-elevated KCs wasn’t changed compared to control lentivirus-treated KCs.3. Methylation modification of Histone H3and acetylated regulation of histone levels after ACTL6a transfectionACTL6a elevation in KCs changed the global protein levels of methylated histones at lysine4,9and79, and the staining patterns of methylated histone3were changed in KCs after ACTL6a elevation; global levels of acetylated-H2a and acetylated-H3were increased in ACTL6a+KCs, accompanied by decreased phosphorylated histone deacetylase (HDAC) level.4. ACTL6a elevation altered cellular differentiation of KCs.ACTL6a elevation in KCs changed the cellular differentiation state, and induced a reries of cellular differention related protein, such as keratin10, keratinl4, involucrin and loricrin.5. ACTL6a elevation altered cellular proliferation, migration, senescence and ROS.Lentivirus-induced ACTL6a overexpression prompted proliferation and migration of normal KCs, and protected normal keratinocytes from cellular senescence and ROS reaction[Conclusions]1. Both ACTL6a and HPly expressed in normal and psoriatic skin. ACTL6a showed similar nuclear staining pattern in normal and psoriatic skin, but HP1γ showed different staining pattern in normal and psoriatic skin.2. Expression of ACTL6a and HPly protein in KCs from peri-lesional skin was higher than KCs from psoriatic lesional skin, non-lesional skin and normal skin.3. Overexpression of ACTL6a prompted tether of HP1γ to the periphery of nuclei.4. ACTL6a involved in the methylated and acetylated regulations of histones within keratinocytes.5、Elevation of ACTL6a in KCs related to celluar differentiation state and altered other cellular functions, including cellular proliferation, migration, senescence and ROS.。6、Expression of ACTL6a in KCs from psoriatic tissues may involve in the pathogenesis of psoriasis.7、It is the limits of our research that we didn’t further explore the exact mechanism of elevated ACTL6a-induced nuclear HPly redistribution, and the genes or signaling pathways they regulated in KCs. |
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