Dataset Information

BMP6 Regulates Corneal Epithelial Cell Stratification by Coordinating Their Proliferation and Differentiation and Is Upregulated in Pterygium.

ABSTRACT: Purpose:Proper balance between cell proliferation and differentiation is essential for corneal epithelial (CE) stratification and homeostasis. Although bone morphogenetic protein-6 (BMP6) is known to be expressed in the CE for over 25 years, its function in this tissue remains unknown. Here, we test the hypothesis that BMP6 promotes CE cell stratification and homeostasis by regulating their proliferation and differentiation. Methods:We employed postnatal day-12 (PN-12), PN-14, PN-20, and PN-90 mouse eyes; human corneal limbal epithelial (HCLE) cells; and ocular surface fibrovascular disease pterygium tissues to evaluate the role of BMP6 in CE proliferation, differentiation, and pathology by RT-qPCR, immunoblots, and/or immunofluorescent staining. Cell proliferation was quantified by immunostaining for Ki67. Results:Coincident with the mouse CE stratification between PN-12 and PN-20, BMP6 was significantly upregulated and the BMP6 antagonist Noggin downregulated. Mature CE retained high BMP6 and low Noggin expression at PN-90. BMP6 and its receptors BMPR1A and BMPR2 were upregulated during in vitro stratification of HCLE cells. Consistent with its anti-proliferative role, exogenous BMP6 suppressed HCLE cell proliferation, downregulated cyclin-D1 and cyclin-D2, and upregulated cell-cycle inhibitors Krüppel-like factor 4 (KLF4) and p21. BMP6 also upregulated the desmosomal cadherins desmoplakin and desmoglein in HCLE cells, consistent with its pro-differentiation role. Human pterygium displayed significant upregulation of BMP6 coupled with downregulation of Noggin and cell-cycle suppressors KLF4 and p21. Conclusions:BMP6 coordinates CE stratification and homeostasis by regulating their proliferation and differentiation. BMP6 is significantly upregulated in human pterygium concurrent with downregulation of Noggin, KLF4, and p21.

SUBMITTER: Tiwari A

PROVIDER: S-EPMC7452852 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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BMP6 Regulates Corneal Epithelial Cell Stratification by Coordinating Their Proliferation and Differentiation and Is Upregulated in Pterygium.

Tiwari Anil A Swamynathan Sudha S Campbell Gregory G Jhanji Vishal V Swamynathan Shivalingappa K SK

Investigative ophthalmology & visual science 20200801 10

<h4>Purpose</h4>Proper balance between cell proliferation and differentiation is essential for corneal epithelial (CE) stratification and homeostasis. Although bone morphogenetic protein-6 (BMP6) is known to be expressed in the CE for over 25 years, its function in this tissue remains unknown. Here, we test the hypothesis that BMP6 promotes CE cell stratification and homeostasis by regulating their proliferation and differentiation.<h4>Methods</h4>We employed postnatal day-12 (PN-12), PN-14, PN- ...[more]

PMID: 32845956

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Project description:BackgroundEpigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.Methodology/principal findingsHuman LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.Conclusion/significanceWe found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.

Project description:PurposeDual specificity phosphatases (DUSPs) modulate the duration and magnitude of phospho-activation of Erk1/2, p38 and JNK1/2, the terminal kinases (TKs) of the mitogen activated protein kinase (MAPK) cascades. Three DUSPs, DUSP1, DUSP5, and DUSP6, are overexpressed in ocular surface side population stem cells (SPSCs). Our objective was to identify the impact of these enzymes on TK phosphorylation and proliferation of corneal epithelial cells.MethodsSV40 immortalized (sv) and expanded fresh human corneal epithelial cells (efHCECs) were transduced with lentivectors to elicit expression of shRNAmir against DUSP1, DUSP5, and JNK1 to thereby create the DUSP1i, DUSP5i and JNKi cell sublines, or overexpress DUSP6 (henceforth DUSP6(+)), respectively. TK phosphorylation status and proliferation rates were determined by immunoblotting and (3)H thymidine uptake.ResultsIn both ef and svHCECs, EGF supplementation after a 24 h serum starvation caused a rapid 5-15 min spike in the phosphorylation of all three TK types. This was followed by gradual decreases to low phosphorylation levels within one h. These declines coincided with dramatic increases in DUSP1 and DUSP5 protein expression. In DUSP1i, the DUSP1 increase was abolished. All 3 TKs maintained high phosphorylation levels for at least 90 min and proliferation rates were unchanged from non-transduced cells. In DUSP5i, the DUSP5 protein increase was prevented, the post peak phosphorylation decrease occurred only on Erk1/2 and the proliferation rate increased by 50%-60%. In JNK1i, JNK1 was essentially knocked out and proliferation rates were also markedly elevated. At steady-state, DUSP1i maintained high levels of pJNK1/2 expression. In DUSP6(+) Erk1/2 phosphorylation was prevented and proliferation rates decreased to less than 50%.ConclusionsDUSP5 and DUSP6 selectively control ERK pathway activity and proliferation. The lack of an effect of DUSP1 knockdown on proliferation can be attributed to its pan-MAPK effect. The expected augmented proliferative response due to enhanced and prolonged phosphorylation of Erk1/2 following DUSP1 knockdown does not occur because a pJNK1/2 antiproliferative effect is simultaneously unleashed.

Dataset Information

BMP6 Regulates Corneal Epithelial Cell Stratification by Coordinating Their Proliferation and Differentiation and Is Upregulated in Pterygium.

Publications

BMP6 Regulates Corneal Epithelial Cell Stratification by Coordinating Their Proliferation and Differentiation and Is Upregulated in Pterygium.

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