Julia Katsnelson

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

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 up-regulated 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.

MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism

Corneal epithelium relies on abundant glycogen stores as its primary energy source. MicroRNA‐31 (miR‐31), a corneal epithelial‐preferred miRNA, negatively regulates factor inhibiting hypoxia‐inducible factor‐1 (FIH‐1). Since HIF‐1α is involved in anaerobic energy production, we investigated the role that miR‐31 and FIH‐1 play in regulating corneal epithelial glycogen. We used antagomirs (antago) to reduce the level of miR‐31 in primary human corneal epithelial keratinocytes (HCEKs), and a miR‐31‐resistant FIH‐1 to increase FIH‐1 levels. Antago‐31 raised FIH‐1 levels and significantly reduced glycogen stores in HCEKs compared to irrelevant‐antago treatment. Similarly, HCEKs retrovirally transduced with a miR‐31‐resistant FIH‐1 had markedly reduced glycogen levels compared with empty vector controls. In addition, we observed no change in a HIF‐1α reporter or known genes downstream of HIF‐1α indicating that the action of FIH‐1 and miR‐31 on glycogen is HIF‐1α‐independent. An enzyme‐dead FIH‐1 mutation failed to restore glycogen stores, indicating that FIH‐1 negatively regulates glycogen in a hydroxylase‐independent manner. FIH‐1 overexpression in HCEKs decreased AKT signaling, activated GSK‐3β, and inactivated glycogen synthase. Treatment of FIH‐1‐transduced HCEKs with either a myristolated Akt or a GSK‐3β inhibitor restored glycogen stores, confirming the direct involvement of Akt/GSK‐3β signaling. Silencing FIH‐1 in HCEKs reversed the observed changes in Akt‐signaling. Glycogen regulation in a HIF‐1α‐independent manner is a novel function for FIH‐1 and provides new insight into how the corneal epithelium regulates its energy requirements.—Peng, H., Hamanaka, R. B., Katsnelson, J., Hao, L., Yang, W., Chandel, N. S., Lavker, R. M. MicroRNA‐31 targets FIH‐1 to positively regulate corneal epithelial glycogen metabolism. FASEB J. 26, 3140–3147 (2012). www.fasebj.org

microRNA-103/107 family regulates multiple epithelial stem cell characteristics

The stem cell niche is thought to affect cell cycle quiescence, proliferative capacity, and communication between stem cells and their neighbors. How these activities are controlled is not completely understood. Here we define a microRNA family (miRs‐103/107) preferentially expressed in the stem cell‐enriched limbal epithelium that regulates and integrates these stem cell characteristics. miRs‐103/107 target the ribosomal kinase p90RSK2, thereby arresting cells in G0/G1 and contributing to a slow‐cycling phenotype. Furthermore, miRs‐103/107 increase the proliferative capacity of keratinocytes by targeting Wnt3a, which enhances Sox9 and YAP1 levels and thus promotes a stem cell phenotype. This miRNA family also regulates keratinocyte cell‐cell communication by targeting: (a) the scaffolding protein NEDD9, preserving E‐cadherin‐mediated cell adhesion; and (b) the tyrosine phosphatase PTPRM, which negatively regulates connexin 43‐based gap junctions. We propose that such regulation of cell communication and adhesion molecules maintains the integrity of the stem cell niche ultimately preserving self‐renewal, a hallmark of epithelial stem cells. Stem Cells 2015;33:1642–1656

MicroRNAs-103/107 coordinately regulate macropinocytosis and autophagy

The miR-103/107 family is preferentially expressed in the stem cell–enriched limbal epithelium and regulates multiple characteristics associated with stem cells. Park et al. show that miR-103/107 also contribute to limbal epithelial homeostasis by suppressing macropinocytosis and preserving end-stage autophagy.

FIH-1/c-kit signaling: a novel contributor to corneal epithelial glycogen metabolism.

PURPOSE Corneal epithelial cells have large stores of glycogen, which serve as their primary energy source. Recently, we demonstrated that factor-inhibiting hypoxia-inducible factor 1 (FIH-1) diminished glycogen stores in vitro and in vivo, working through the Akt/Glycogen Synthase Kinase (GSK)-3β pathway. In this study we investigated the relationship between FIH-1 and c-kit as it pertains to limbal and corneal epithelial glycogen stores. METHODS Limbal and corneal epithelia from wild-type FIH-1(-/-) and Kit(W/Wv) mice were stained with periodic acid Schiff (PAS) to detect glycogen. RNA samples prepared from laser-capture microdissected populations of limbal epithelium were subjected to real-time quantitative PCR to determine c-kit ligand expression. Submerged cultures of primary human corneal epithelial keratinocytes (HCEKs) transduced with FIH-1 were treated with c-kit ligand to establish further a FIH-1/c-kit interaction via Western analysis. Akt phosphorylation was assessed by Western blotting. RESULTS The limbal epithelial cells of FIH-1 null mice had an increase in glycogen levels as well as increased c-kit ligand mRNA compared with wild-type controls. Consistent with a FIH-1/c-kit association, the diminished Akt signaling observed in FIH-1-overexpressing HCEKs could be restored by the addition of c-kit ligand. Interestingly, Akt signaling and glycogen content of the corneal epithelium were significantly decreased in c-kit mutant mice. CONCLUSIONS c-Kit signaling has been shown to affect glucose metabolism via the Akt/GSK-3β pathway. An inverse relationship between FIH-1 and c-kit signaling pathways accounts, in part, for differences in glycogen content between corneal and limbal epithelial cells.

MicroRNAs Enhance Keratinocyte Proliferative Capacity in a Stem Cell-Enriched Epithelium

MicroRNAs are critical regulators of stem cell behavior. The miR-103/107 family is preferentially expressed in the stem cell-enriched corneal limbal epithelium and plays an important role in coordinating several intrinsic characteristics of limbal epithelial stem cells. To elucidate further the mechanisms by which miRs-103/107 function in regulating limbal epithelial stem cells, we investigate the global effects of miRs-103/107 on gene expression in an unbiased manner. Using antagomirs-103/107, we knocked down endogenous miRs-103/107 in keratinocytes and conducted an mRNA profiling study. We show that miRs-103/107 target mitogen-activated protein kinase kinase kinase 7 (MAP3K7) and thereby negatively regulate the p38/AP-1 pathway, which directs epithelial cells towards a differentiated state. Pharmacological inhibition of p38 increases holoclone colony formation, a measure of proliferative capacity. This suggests that the negative regulation of p38 by miRs-103/107 contributes to enhanced proliferative capacity, which is a hallmark of stem cells. Since miRs-103/107 also promote increased holoclone colony formation by regulating JNK activation through non-canonical Wnt signaling, we believe that this microRNA family preserves “stemness” by mediating the crosstalk between the Wnt/JNK and MAP3K7/p38/AP-1 pathways.