Purpose:
The cornea stroma consists of collagen fibrils that exist in a highly ordered structure, however the mechanisms by which collagen expression and its complex architecture are regulated are not well understood. Insight into the mechanisms that underlie collagen expression and fibril architecture would shed light onto the etiologies of corneal diseases. Thus, identifying specific pathways or proteins that are in involved in stromal collagen regulation could help better explain how collagen is regulated in health, and what goes wrong in cases of pathology. A protein recently shown to induce collagen expression is the cytoplasmic protein called Shroom3. This F-actin and Rho-kinase binding protein is best known for regulating epithelial cell shape during embryonic morphogenesis, but the mechanisms by which it regulates collagen expression have not been widely studied. Previous research has suggested that abnormal SHROOM3 disrupts cornea development in mouse embryos and that a homozygous missense mutation in this gene has been associated with the stromal thinning disease known as keratoconus. Therefore, the purpose of this research was to further characterize the corneal phenotype in Shroom3-deficient embryos and discern what effect the keratoconus-associated mutation has on Shroom3 function.
Methods:
Embryonic corneal Shroom3 expression was observed using X-gal labeling of Shroom3+/- embryos that express X-galactosidase under the control of the endogenous Shroom3 promoter. To determine the function of Shroom3 during cornea development, control and Shroom3-deficient mouse embryos were analyzed using histological staining, immunofluorescent imaging, and electron microscopy to ascertain the effect on collagen expression, fibril size, keratocyte number, stromal thickness and corneal epithelial shape. The consequences of mutations on Shroom3 function were analyzed in kidney- (MDCK) and cornea-derived (SIRC) cells following transfection with plasmids expressing wild-type Shroom3 (Shroom3wt), a Rho-kinase binding deficient mutation (Shroom3R1838C) and the keratoconus-associated mutation (Shroom3G59V). Epithelial cell shape and collagen expression were analyzed by immunofluorescent imaging and RT-PCR analysis, and electron microscopy was performed to analyze the effect Shroom3 has on collagen expression, fibril size, and corneal phenotypes.
Results:
Shroom3 expression in the cornea was observed in the central and peripheral cornea, however expression was higher in the periphery and posterior stroma. Shroom3-/- cornea epithelial cells had larger apical areas compared to Shroom3+/- cells, suggesting reduced apical constriction. Shroom3-/- corneas displayed a reduction in total number of keratocytes in the central cornea. There was no difference in stromal thickness between Shroom3+/- and Shroom3-/- corneas when measured directly, but there was a reduction in the layers of keratocytes observed in Shroom3-/- corneas which acted as an indirect measure of stromal thickness. The intensity of Col1 staining was reduced in the posterior stroma versus the anterior stroma in Shroom3-/- corneas, and the intensity of the Shroom3 null posterior stroma was lower than that of the Shroom3+/- posterior stroma. Shroom3R1838C and Shroom3G59V variants caused increased Col4a1 expression compared to the Shroom3wt, and decreased Col1a1 expression, whereas the opposite was observed with in the Shroom3wt samples. The average stromal collagen fibril diameter was larger in Shroom3-/- corneas compared to Shroom3+/- corneas.
Conclusion:
Shroom3 is required for normal corneal development. Specifically, these results suggest that Shroom3 is responsible for facilitating lens vesicle separation from the overlying ectoderm by regulating corneal epithelial cell shape. Additionally, these data suggest Shroom3 normally promotes stromal expression of collagen type I and inhibits collagen type IV, limits fibril diameter size, and influences keratocyte number. Because both the G59V and R1838C mutations to Shroom3 disrupt its collagen regulation function, it further suggests that Shroom3 relies on both its PDZ and Rho-kinase-binding domains to perform this function. Future studies exploring the mechanisms underlying Shroom3-dependent corneal collagen regulation and development may lead to a better understanding of the pathologies that disrupt collagen and lead to corneal disorders such as keratoconus.