Abstract
BACKGROUND: HC-HA/PTX3 (a complex formed by high molecular weight hyaluronan covalently linked to heavy chain 1 of inter-α-trypsin inhibitor and tightly bound to pentraxin 3) is a unique extracellular matrix from human amniotic membrane that exerts an anti-scarring action and reprograms human corneal fibroblasts (HCF) and myofibroblasts to corneal stromal keratocytes in the absence of transforming growth factor β1 (TGFβ1) by downregulating canonical Smad-mediated signaling and upregulating bone morphogenetic protein (BMP) signaling. It remains unclear whether HC-HA/PTX3 can further reprogram HCF into neural crest (NC) progenitors in the presence of TGFβ1. METHODS: Human corneal fibroblasts were seeded on plastic, immobilized hyaluronic acid (HA) or HC-HA/PTX3 or on plastic with or without soluble HA and HC-HA/PTX3 in DMEM + 10% fetal bovine serum (FBS), with or without various inhibitors with or without TGFβ1. Transcript expression of NC and signaling markers was determined by RT-qPCR. Immunostaining was performed to monitor cytolocalization of signaling markers and α-smooth muscle actin (α-SMA). Raft separation before Western blot was used to study protein distributions in both rafts. Western blot and ELISA were used to measure relative protein level. RESULTS: Herein, we show for the first time that in the presence of exogenous TGFβ1, HC-HA/PTX3 continues to reprogram HCF into NC progenitors by upregulating mRNA expression of NC markers, confirmed by successful induction into human corneal endothelial cells, highlighted by hexagonal shape, mRNA expression of corneal endothelial markers and junctional staining of corneal endothelial markers such as Na-K-ATPase, α-catenin, β-catenin, F-actin, N-cadherin, p120 and ZO-1 but the lack of fibrogenic marker S100A4. Such reprogramming requires suppression of TGFβ1 SMAD-mediated canonical signaling that starts from HC-HA/PTX3 binding with CD44 to sequester type II TGFβ receptor (TβRII) in lipid raft and ends with downregulation of TβRII by nuclear translocation of cyclin D1. Mechanistically, nuclear translocation of cyclin D1 is mediated by activation of transforming growth factor-beta-activated kinase 1-transcription factor Jun (TAK1-cJUN) noncanonical signaling because of type I TGFβ receptor (TβRI) and type III TGFβ receptor (TβRIII) (without TβRII) in non-lipid raft as well as by nuclear translocation of CD44 intracellular domain (CD44ICD) formed by Membrane Type 1 Matrix Metalloproteinase (MT1MMP)/γ-secretase cleavage to facilitate such reprogramming. CONCLUSIONS: Thus, HC-HA/PTX3 from amniotic membrane can be deployed as a new strategy to reverse scar toward regeneration.