Yujin Zhang

Lumican Binds ALK5 to Promote Epithelium Wound Healing

Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-β receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC13 peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.

Mastermind-like transcriptional co-activator-mediated Notch signaling is indispensable for maintaining conjunctival epithelial identity

Conjunctival goblet cells primarily synthesize mucins to lubricate the ocular surface, which is essential for normal vision. Notch signaling has been known to associate with goblet cell differentiation in intestinal and respiratory tracts, but its function in ocular surface has yet to be fully characterized. Herein, we demonstrate that conditional inhibition of canonical Notch signaling by expressing dominant negative mastermind-like 1 (dnMaml1) in ocular surface epithelia resulted in complete suppression of goblet cell differentiation during and subsequent to development. When compared with the ocular surface of wild-type mice (OSWt), expression of dnMaml1 at the ocular surface (OSdnMaml1) caused conjunctival epithelial hyperplasia, aberrant desquamation, failure of Mucin 5ac (Muc5ac) synthesis, subconjunctival inflammation and epidermal metaplasia in cornea. In addition, conditional deletion of Notch1 from the ocular surface epithelia partially recapitulated OSdnMaml1 phenotypes. We have demonstrated that N1-ICD (Notch1 intracellular domain) transactivated the mouse Krüppel-like factor 4 (Klf) promoter and that Klf4 directly bound to and significantly potentiated the Muc5ac promoter. By contrast, OSdnMaml1 dampened Klf4 and Klf5 expression, and diminished Muc5ac synthesis. Collectively, these findings indicated that Maml-mediated Notch signaling plays a pivotal role in the initiation and maintenance of goblet cell differentiation for normal ocular surface morphogenesis and homeostasis through regulation of Klf4 and Klf5.

Dexamethasone induces cross-linked actin networks in trabecular meshwork cells through noncanonical wnt signaling.

PURPOSE Dexamethasone (DEX) regulates aqueous humor outflow by inducing a reorganization of the cytoskeleton to form cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells. Rho-associated protein kinase (ROCK) has been demonstrated to have an important role in this process, but the upstream components leading to its activation remain elusive. The purpose of the study is to demonstrate that noncanonical Wnt signaling mediates the DEX-induced CLAN formation in TM cells. METHODS The TM cells were treated with 100 nM DEX in low serum medium for over 7 days. The medium was changed every 3 days. The cells were harvested and subjected to molecular analysis for the expression of Wnt ligands. Stress fiber structures were revealed by Phalloidin staining. Lentivirus-based shRNA against noncanonical Wnt receptor (Ror2) was used to determine the role of noncanonical Wnt signaling in DEX-induced CLAN formation. RESULTS The DEX induced stress fiber rearrangement in TM cells. A noncanonical Wnt ligand (Wnt5a) was upregulated by DEX as demonstrated by Wnt ligand degenerate PCR, real-time quantitative PCR (qRT-PCR), and Western blotting. Knocking-down Ror2, the receptor of noncanonical Wnt signaling, abolished the effects of DEX on the TM cells. CONCLUSIONS Our data suggest that DEX induces the upregulation of noncanonical Wnt ligand Wnt5a. Recombinant WNT5a protein induces CLAN formation through the noncanonical Wnt receptor ROR2/RhoA/ROCK signaling axis. Given the similarities between DEX-induced ocular hypertension and primary open-angle glaucoma, our results provide a mechanism of action for applying ROCK inhibitor to treat primary open-angle glaucoma.

Aberrant expression of a β-catenin gain-of-function mutant induces hyperplastic transformation in the mouse cornea

β-catenin signaling has been shown to play a fundamental role in embryonic development and tumorigenesis. In this study, we investigated the role of β-catenin (Ctnnb1) in corneal homeostasis and tumorigenesis. Conditional expression of a murine Ctnnb1 gain-of-function mutation alone caused corneal neoplasia and neovascularization, resembling human ocular surface squamous neoplasia (OSSN). These corneas displayed an upregulation of cell proliferative markers (PCNA and p63), while presenting downregulation of both the Pax-6 transcription factor and the corneal differentiation marker cytokeratin 12. In addition, the expression of limbal-type keratin 15 ectopically extended to cornea, but the pattern of conjunctival keratin 4 and epidermal keratin 10 were unchanged. Moreover, epithelial E-cadherin and laminins decreased concomitantly with elevated levels of MMP-7. We also noticed a dramatic upregulation of pro-angiogenic factors (Vegf-A, Vegfr1) and angiopoietins in these corneas. Interestingly, all human OSSN specimens examined revealed nuclear β-catenin immunoreactivity. Taken together, these results argue that β-catenin activation is a crucial step during OSSN pathogenesis. Thus, inhibition of β-catenin might be beneficial for treating this disease.

Notch gain of function in mouse periocular mesenchyme downregulates FoxL2 and impairs eyelid levator muscle formation, leading to congenital blepharophimosis

Notch signaling is pivotal for the morphogenesis and homeostasis of many tissues. We found that aberrant Notch activation in mouse neural-crest-derived periocular mesenchymal cells (POMCs), which contribute to the formation of corneal and eyelid stroma, results in blepharophimosis. Compound transgenic mice overexpressing the Notch1 intracellular domain (N1-ICD) in POMCs (POMCN1-ICD) showed relatively minor effects on the cornea, but increased cell apoptosis and decreased cell proliferation during eyelid morphogenesis. Eyelid closure at E15.5 and eyelid formation at birth were incomplete. In further analyses, overexpression of N1-ICD impaired eyelid levator smooth muscle formation by downregulating the transcription factor FoxL2. This is similar to the effect of haploinsufficiency of FOXL2 in humans, which results in type II BPES (blepharophimosis, ptosis and epicanthus inversus syndrome). In vitro studies showed that FoxL2 expression is augmented by a low dose of N1-ICD but was downregulated by a high dose, depending on the extent of Hes-1 and Hey-1 activation. Moreover, transfection of CMV-FoxL2 enhanced α-SMA promoter activity. These data strongly imply that a physiologically low level of Notch1 is crucial for proper FoxL2 expression in POMCs, which is, in turn, essential for Müeller muscle formation and normal eyelid development.

Role of SH2-containing tyrosine phosphatase Shp2 in mouse corneal epithelial stratification.

PURPOSE Shp2 protein tyrosine phosphatase mediates a wide variety of receptor tyrosine kinases (RTK) cell signaling. Herein, we investigate the role of Shp2 in corneal morphogenesis and homeostasis. METHODS Shp2 was conditionally knocked out (Shp2(cko)) in Krt14-rtTA;tet-O-Cre;Shp2(f/f) triple transgenic mice administrated with doxycycline (Dox) from postnatal day 1 (P1) to P10, P15, and P25, respectively. In addition, corneal epithelial debridement was performed in adult (P42) mice treated with or without Dox for 8 days (from P42-P50). Mouse eyes were then subjected to histology and immunohistochemistry. RESULTS Shp2(cko) revealed impaired stratification of conjunctival and corneal epithelia during morphogenesis. Likewise, Shp2(cko) failed to restore epithelial stratification after a corneal epithelial wound in adult Shp2(cko). At the cellular level, the ratio of proliferating cell nuclear antigen (PCNA-positive)/total basal cells remained unchanged, but cells in G2/M (survivin-positive) phase was significantly increased in Shp2(cko) as compared with those in the control littermate. Interestingly, deltaN-p63 (ΔNp63) expression and the asymmetric division of the basal cells were coincidentally dampened in Shp2(cko). Transmission electron microscopic study showed that desmosome and hemidesmosome densities were reduced in the corneal epithelium of Shp2(cko). Immunohistochemistry also demonstrated that expression of E-cadherin/β-catenin junction and laminin-β1 was extensively downregulated in Shp2(cko). On the other hand, corneal epithelium lacking Shp2 remained positive for K14, Pax-6, and keratin 12 (K12), suggesting that Shp2 was dispensable for the corneal epithelial-type differentiation. CONCLUSIONS These data argued that Shp2 deficiency predominantly impacted p63-dependent cell division and cell adhesive ability, which resulted in the impairment of stratification during corneal epithelial development and wound healing.

Perturbed meibomian gland and tarsal plate morphogenesis by excess TGFα in eyelid stroma

Transforming growth factor alpha (TGFα) belongs to the epidermal growth factor (EGF) family and is known to play an important role during eyelid morphogenesis. In this study, we showed that ectopic expression of TGFα in the stroma of Kera-rtTA/tet-O-TGFα bitransgenic mice results in precocious eye opening, abnormal morphogenesis of the meibomian gland, tendon and tarsal plate malformation and epithelium hyperplasia. TGFα did not change proliferation and differentiation of meibocytes, but promoted proliferation and inhibited differentiation of the tarsal plate tenocytes. These results suggest that proper formation of the tendon and tarsal plate in the mouse eyelid is required for normal morphogenesis of the meibomian gland.