microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation

Abstract

Notch plays a critical role in the transition from proliferation to differentiation in the epidermis and corneal epithelium. Furthermore, aberrant Notch signaling is a feature of diseases like psoriasis, eczema, nonmelanoma skin cancer, and melanoma where differentiation and proliferation are impaired. Whereas much is known about the downstream events following Notch signaling, factors responsible for negatively regulating Notch receptor signaling after ligand activation are incompletely understood. Notch can undergo hydroxylation by factor-inhibiting hypoxia-inducible factor 1 (FIH-1); however, the biological significance of this phenomenon is unclear. Here we show that FIH-1 expression is upregulated in diseased epidermis and corneal epithelium. Elevating FIH-1 levels in primary human epidermal keratinocytes (HEKs) and human corneal epithelial keratinocytes (HCEKs) impairs differentiation in submerged cultures and in a "three-dimensional" organotypic raft model of human epidermis, in part, via a coordinate decrease in Notch signaling. Knockdown of FIH-1 enhances keratinocyte differentiation. Loss of FIH-1 in vivo increased Notch activity in the limbal epithelium, resulting in a more differentiated phenotype. microRNA-31 (miR-31) is an endogenous negative regulator of FIH-1 expression that results in keratinocyte differentiation, mediated by Notch activation. Ectopically expressing miR-31 in an undifferentiated corneal epithelial cell line promotes differentiation and recapitulates a corneal epithelium in a three-dimensional raft culture model. Our results define a previously unknown mechanism for keratinocyte fate decisions where Notch signaling potential is, in part, controlled through a miR-31/FIH-1 nexus.

FIH-1/c-Kit Signaling: A Novel Contributor to Corneal Epithelial Glycogen Metabolism

“…Limbal epithelium from 5-lm frozen sections was isolated and captured using a PALM lasercapture system (Carl Zeiss) as previously described. 24 …”

“…HCEKs transduced with retroviral supernatants carrying either LZRS-empty vector or FIH-1 24 with or without SCF were lysed with radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.25% deoxycholic acid, 1% Tergitol Nonidet P-40 (NP-40), 1 mM EDTA, 2 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride, phosphatase inhibitor cocktail (Thermo Scientific, West Palm Beach, FL), and protease inhibitor cocktail (Thermo Scientific), followed by centrifugation at 15,000g for 15 minutes. Mouse corneal epithelia were peeled away from the underlying stroma by incubating whole globes with PBS containing 20 mM EDTA for 1 hour at 378C 25 and subsequently were lysed with 8 lM urea sample buffer as previously described.…”

“…26 Western blots were performed as described previously. 24 The following antibodies were used: p-Akt (S473), p-Akt (T308), p-GSK-3b, p-glycogen synthase, p-mammalian target of rapamycin (mTOR) p-4E-BP1 (Cell Signaling Technologies), FIH-1, GSK3b, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology).…”

“…24 For inducing c-kit signaling, HCEKs were treated with 100 ng/mL SCF (Cell Signaling Technologies, Danvers, MA) for 24 hours.…”

Noncoding RNAs and Their Potential Therapeutic Applications in Tissue Engineering

“…Inhibit proliferation and migration miR-182 [54]; let-7 [55]; miR-1 [56] Promote proliferation and migration miR-221, miR-222 [57] Cell type and function ncRNAs Inhibit migration miR-9 [58] Promote migration miR-132 [59] Regulate dedifferentiation and proliferation miR-34a [60] Regulate myelination miR-140 [60]; miR-29a [61] Regulate fibrinolysis miR-340 [ [83][84][85][86] miR-27b, miR-224 [87]; miR-574-3p, miR-31 [88] Inhibit proliferation miR-34, siRNA-P63 [89]; miR-720 [90]; miR-210, siRNA-E 2 F 3 [91] Improve proliferation, reduce differentiation miR-125b, siRNA-FGFR 2 [92,93] Inhibit proliferation, induce differentiation miR-24, siRNA-PAK4 [94] Keratinocytes Improve migration miR-205 [95][96][97] Inhibit migration, improve proliferation miR-483-3p [98] Inhibit migration miR-198 [99] Improve migration, inhibit migration miR-21 [100,101] Improve migration and proliferation miR-31, siRNA-EMP-1, siRNA-TGF-β [102] Regulate apoptosis lncRNA-p21 [103] Fibroblasts Inhibit proliferation let-7, miR-125 [104] Improve proliferation miR-29 [104]; miR-21 [105]; miR-22 [106]; lncRNA-H19 [107] Induce senescence miR-152, miR-181a [108]; miR-141 [109]; miR-143 [110]; miR-519a [11...…”