Abstract
Purpose :
The corneal epithelial (CE) homeostasis depends on a delicate balance between their proliferation and differentiation. Though the CE functions of transforming growth factor (TGF)-β1 and -β2 have been studied extensively, the same is not true for the other members of the TGF superfamily including bone morphogenetic protein-6 (BMP6). Here we test the hypothesis that BMP6 contributes to CE homeostasis by regulating their proliferation and differentiation.
Methods :
We employed mouse pups between post-natal day-12 (PN12) and PN20, human corneal limbal epithelial (HCLE) cells and human pterygium samples to study the role of BMP6 in CE proliferation, differentiation and pathology. HCLE cells were maintained and treated with BMP6 (50ng/ml) in Keratinocyte-Serum Free Medium (K-SFM), and stratified in DMEM/F12, 10% FCS and 10 ng/ml EGF. Selected BMP6-target gene expression was evaluated by QPCR, immunoblots and/or immunofluorescent staining. Cell proliferation was assayed by cell titer blue and immunostaining for Ki67.
Results :
Coincident with the mouse CE stratification, Bmp6 expression increased gradually between PN12-PN14 (3.7-fold), culminating with robust expression at PN20 (183.4-fold). Similar increase was observed in BMP6 transcripts (101-fold) and protein (9.8-fold) upon in vitro stratification of HCLE cells. QPCR revealed an upregulation of BMP receptors BMPR1A and BMPR2 (1.9- and 3.1-fold, respectively), KLF4 (3.4-fold), and CE markers DSG and DSP (both 6.5-fold) upon stratification. Addition of exogenous BMP6 to HCLE cells resulted in a significantly decreased cell proliferation, flattened cellular morphology coupled with the appearance of multi-cell layers reminiscent of stratification. BMP6 also downregulated the expression of cyclin-D1 (to 0.8-fold) and -D2 (to 0.5-fold), while upregulating KLF4 (2.2-fold), p21 (2.3-fold), CE markers DSP and DSG (1.9- and 4.6-fold, respectively), and sequestering β-catenin in cell membranes. Moreover, BMP6 was significantly upregulated (6.38-fold) in human pterygium samples compared with the normal tissue.
Conclusions :
By demonstrating that BMP6 i) is upregulated during CE stratification, ii) inhibits proliferation and stimulates differentiation of HCLE cells and iii) is overexpressed in pterygium, these results provide evidence of a role for BMP6 in CE stratification and pathology.
This is a 2020 ARVO Annual Meeting abstract.