Forkhead box-O1 (FoxO1) is a key nutrient- and growth factor-dependent regulator of metabolism, but its functional role in human primary keratinocytes (HPKs) is less known. To investigate the role of FoxO1 in HPKs and effect of insulin-like growth factor 1 (IGF-1) and isotretinoin on FoxO1 expression, HPKs were treated with 1.2 mmol/L calcium chloride, 1-20 ng/mL IGF-1 and 0.1-10 μmol/L isotretinoin. Recombinant adenovirus expressing FoxO1 or FKHR shRNA lentivirus transfection was introduced to upregulate or silence FoxO1 expression. Epidermal FoxO1 immunostaining was lower in acne lesion than in normal skin. FoxO1 overexpression induced involucrin expression, G2/M arrest and apoptosis but suppressed proliferation, while FoxO1 silencing decreased involucrin expression but increased proliferation, S phase and viable cells in HPKs. IGF-1 downregulated FoxO1 and involucrin but upregulated p-Akt expression in HPKs, which was blocked by pretreatment with LY294002. Isotretinoin enhanced FoxO1, p53 and p21 but inhibited p-FoxO1 and involucrin expression in HPKs. These results demonstrate that FoxO1 promotes differentiation and apoptosis in HPKs. IGF-1 may reduce keratinocyte differentiation through PI3K/Akt/FoxO1 pathway, while isotretinoin can reinforce FoxO1 expression. FoxO1 may be involved in acne pathogenesis and could serve as a potential therapeutic target.

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Sebocyte differentiation is a continuous process, but its potential molecular mechanism remains unclear. We aimed to establish a novel sebocyte differentiation model using human primary sebocytes and to identify the expression profiles of differentiation-associated proteins. Primary human sebocytes were cultured on Sebomed medium supplemented with 2% serum for 7 days. Flow cytometry showed that S phase cells were decreased time-dependently, while G1 and subG1 (apoptosis) phase cells increased under serum starvation. Transmission electron microscopy and Oil Red O staining revealed a gradual increase of intracellular lipid accumulation. Expression of proliferation marker was diminished, while expression of differentiation, apoptosis, and lipogenic markers elevated gradually during 7-day culture. iTRAQ analysis identified 3582 expressed proteins in this differentiation model. Compared with day 0, number of differentially expressed proteins was 132, 54, 321, and 96 at days 1, 3, 5, and 7, respectively. Two overexpressed proteins (S100 calcium binding protein P and ferredoxin reductase) and 2 downexpressed proteins (adenosine deaminase and keratin 10) were further confirmed by Western blot and immunohistochemistry.

Abstract. Endoribonuclease (NendoU) is conserved and plays essential roles in viral life cycle among all nidoviruses. Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 11 (nsp11) is shown to have NendoU activity and its multifunctions have been demonstrated, such as processing RNA, suppressing innate immunity system of infected hosts and regulating the progression of S phase cells. We solve the crystal structure of PRRSV nsp11 mutant (K170A), and the overall structure is greatly different from severe acute respiratory syndrome coronaviruses (SARS-CoV) nsp15, the counterpart of nsp11 in coronaviruses. Compared with SARS-CoV nsp15 which has three domains, PRRSV nsp11 only have two domains: the N-terminal domain (NTD) and C-terminal domain (CTD). Smallangle X-ray scattering data shows that the PRRSV nsp11 has several kinds of oligomeric conformations in solution, this may be due to the missing domain, which mediates the hexamerization of SARS-CoV nsp15. The active center of NendoU is located in the CTD, where the conserved catalytic residues form a positively charged groove and bind the negatively charged RNA. The catalytic regions have several conformations, which are quite diverse. The NTD shares similar structural element with ubiquitin binding protein, but nsp11 cannot bind to ubiquitin according to our data. The flexible catalytic region of nidoviruses endoribonuclease confers an excellent target for drug design. These structural and functional information of PRRSV nsp11 would lay a foundation for better understanding of the molecular mechanism and antiviral drug development.