Research

microscope

Ocular Surface Functions of KLF4 and KLF5

Corneal epithelium (CE) is a self-regenerating stratified squamous tissue that protects the rest of the eye by serving as the anterior-most barrier. Previously, we demonstrated that the Krüppel-like factors KLF4 and KLF5, two of the most abundant transcription factors in the CE, play crucial roles in its maturation and maintenance. We employed spatiotemporally regulated ablation of Klf4 in the adult mouse CE to discover that Klf4, abundantly expressed in the suprabasal cells, is a pro-differentiation factor that suppresses epithelial-mesenchymal transition (EMT) and promotes CE features, regulates CE cell cycle progression by suppressing TGF-b signaling, and coordinates CE cell apical-basal polarity (ABP) and plane of division. Similar studies with conditional ablation of Klf5 in the adult mice revealed that Klf5, abundantly expressed in the basal cells, is a pro-proliferation factor. These results are consistent with a functional dichotomy between CE functions of Klf4 and Klf5 wherein Klf4 promotes superficial cell differentiation while Klf5 promotes basal cell proliferation. Ongoing work in our lab employs innovative mouse genetics, cellular and molecular biological techniques to test if the choice between corneal epithelial cell (CEC) proliferation and differentiation is determined by the delicate balance between the anti- and pro-proliferative activities of Klf4 and Klf5, which bind distinctly configured cis-elements in their target gene promoters in spite of possessing similar DNA-binding domain.

This research is supported by NIH RO1 EY 026533 (PI: Swamynathan)

 

 

Test tubes

Ocular Surface Functions of SLURP1

Though ocular surface inflammatory disorders are among the most common reasons for outpatient visits to an ophthalmologist, the persistent gap in our understanding of immunomodulation remains a barrier for developing relevant new therapies. Corneal immune-privilege is regulated by a redundant set of molecules. Previously, we reported that the secreted Ly6/uPAR related protein-1 (SLURP1): (i) is highly expressed in the corneal epithelium (CE) and is downregulated in pro-inflammatory conditions; (ii) acts as a soluble scavenger of urokinase (uPA); (iii) inhibits human umbilical vein endothelial cell (HUVEC) tube formation; (iv) suppresses neutrophil chemotaxis and transmigration; and (v) suppresses TNF-a-induced cytokine production and stabilizes epithelial cell junctions. Collectively, these studies identified SLURP1 as a potential diagnostic and therapeutic target for inflammatory disorders of the ocular surface and offered glimpses of the underlying mechanisms. We also found that the Slurp1-null mouse (Slurp1X-/-) corneas display dense neovascularization and excessive neutrophil influx five days after silver nitrate cautery. RNA-Seq analysis of the naïve Slurp1X-/- corneal transcriptome predicted key activators of angiogenic inflammation including TGF-b and NFkB pathway components to be upregulated in the absence of Slurp1. Ongoing work in our lab is designed to build on these salient findings by testing if SLURP1 suppresses corneal angiogenic inflammation and neutrophil recruitment by regulating the TGF-b- and uPA-activities that promote NFkB-mediated production of pro-inflammatory molecules.

This research is supported by NIH RO1 EY 031684 (PI: Swamynathan)

Lab bench

Polarity in Ocular Epithelial Cells

Epithelial cells that line the body's outer surface and internal organs are polarized, with well-defined basal and apical sides. Within the eye, corneal epithelium (CE) and retinal pigmented epithelium (RPE) are two such specialized epithelia that play essential roles in ensuring proper vision. We are studying the role of Pard3, a tight junction component and a key determinant of epithelial apicobasal polarity in these tissues.

The CE, a stratified squamous tissue comprised of polarized cells, is essential for maintenance of the corneal transparence. We have developed in vitro cultured human hTC-Epi cell model with CRISPR-mediated mutation in PARD3, and a mouse model with CE-specific ablation of Pard3 to investigate the role of Pard3 in CE structure and function. Ongoing work in our lab is focused on using these model systems to evaluate the role of PARD3 in CE polarity and genetic stability.

The RPE, a monolayer of polarized cells, is essential for maintenance of the neural retina. We have developed in vitro cultured human ARPE-19 cell model with CRISPR-mediated mutation in PARD3, and a mouse model with RPE-specific ablation of Pard3 to investigate the role of Pard3 in RPE structure and function. Preliminary results using these model systems suggest that the RPE lacking Pard3 is defective, suggesting that Pard3 plays a key role in the RPE. Ongoing work in our lab is focused on further evaluating the contributions of polarity on RPE functions and the role of Pard3 in these functions.

This research is supported by Startup Funds from the Department of Ophthalmology, MCOM, USF, Tampa, FL. (PI: Swamynathan)

Recent Publications

Please follow this link for my current bibliography on NCBI: https://www.ncbi.nlm.nih.gov/myncbi/collections/mybibliography/

  1. Swamynathan, S.K. *, Katz, J.P., Kaestner, K.H., Ashery-Padan, R., Crawford, M.A. and Piatigorsky, J. 2007. Conditional deletion of the mouse Klf4 gene results in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells. Mol. Cell. Biol. 27: 182-194. PMID: 17060454.
  2. Swamynathan, S.K. and Piatigorsky, J. 2007. Regulation of the mouse aB-Crystallin and MKBP/HspB2 Promoter activities by shared and gene specific intergenic elements: The importance of context dependency. Int. J. Dev. Biol. 51: 689-700. PMID: 17939115.
  3. Kozmik, Z., Swamynathan, S.K., Ruzickova, J., Jonasova, K., Paces, C., Vlcek, C., and Piatigorsky, J. 2008. Cubozoan crystallins: Evidence for convergent evolution of Pax regulatory sequences. Evol. Dev. 10: 52-61. PMID: 18184357.
  4. Swamynathan, S.K*., Davis, J. and Piatigorsky, J. 2008. Identification of candidate KLF4 target genes reveals the molecular basis of the diverse regulatory roles of KLF4 in the mouse cornea. Invest. Ophthalmol. Vis. Sci. 49: 3360-3370. PMID: 18469187.
  5. Young, R.D., Swamynathan, S.K.*, Boote, C., Mann, M., Quantock, A.J., Piatigorsky, J., Funderburgh, J.L., and Meek, K.M. 2009. Stromal edema in Klf4 conditional null mouse cornea is associated with altered collagen fibril organization and reduced proteoglycans. Invest. Ophthalmol. Vis. Sci. 50:4155-4161. PMID: 19387067.
  6. Swamynathan, S., Kenchegowda, D., Piatigorsky, J. and Swamynathan, S.K. * 2011. Regulation of the corneal epithelial barrier function by Krüppel-like transcription factor 4. Invest. Ophthalmol. Vis. Sci. 52:1762-1769. PMID: 21051695.
  7. Gupta, D., Harvey, S.A., Kaminski, N. and Swamynathan, S.K. * 2011. Mouse conjunctival forniceal gene expression during postnatal development and its regulation by Krüppel-like factor 4. Invest. Ophthalmol. Vis. Sci. 52:4951-4962. PMID: 21398290.
  8. Kenchegowda, D, Swamynathan, S, Gupta, D, Wan, H, Whitsett, J, and Swamynathan S.K*. 2011. Conditional disruption of mouse Klf5 results in defective eyelids with malformed meibomian glands, abnormal cornea and loss of conjunctival goblet cells. Dev. Biol. 356:5-18. PMID: 21600198.
  9. Kenchegowda, D., Harvey, S.A.K., Swamynathan, S., Lathrop, K.L. and Swamynathan, S.K.* 2012. Critical Role of Klf5 in Regulating Gene Expression during Post-Eyelid Opening Maturation of Mouse Corneas. PLoS ONE 7(9): e44771. doi:10.1371/journal.pone.0044771. PMID: 23024760.
  10. Swamynathan, S., Buela, K.A, Kinchington, P., Misawa, H., Lathrop, K.L., Hendricks, R.L. and Swamynathan, S.K.* 2012. Klf4 regulates the expression of Slurp1, which functions as an immunomodulatory peptide in the mouse cornea. Invest. Ophthalmol. Vis. Sci. 53:8433-8446. PMID: 23139280.
  11. Gupta, D., Harvey, S.A., Kenchegowda, D., Swamynathan, S., and Swamynathan, S.K. * 2013. Regulation of mouse lens maturation and gene expression by Krüppel-like factor 4. Exp. Eye Res. 116: 205-218. PMID: 24076321.
  12. Swamynathan, S. and Swamynathan, S.K.* 2014. SLURP-1 modulates corneal homeostasis by serving as a soluble scavenger of urokinase-type plasminogen activator uPA. Invest. Ophthalmol. Vis. Sci. 55:6251-6261. PMID: 25168896.
  13. Delp, E.E., Swamynathan, S., Kao, W.W. and Swamynathan, S.K.* 2015. Spatiotemporally regulated ablation of Klf4 in adult mouse corneal epithelial cells results in altered epithelial cell identity and disrupted homeostasis. Invest. Ophthalmol. Vis. Sci. 56:3549-3558. PMID: 26047041
  14. Swamynathan, S., Delp, E.E., Harvey, S.A.K., Loughner, C.L., Raju, L. and Swamynathan, S.K.* 2015. Corneal expression of SLURP-1 by age, sex, genetic strain and ocular surface health. Invest. Ophthalmol. Vis. Sci. 56:7888-7896. PMID: 26670825.
  15. Swamynathan, S., Loughner, C.L. and Swamynathan, S.K.* 2017. Inhibition of HUVEC tube formation via suppression of NFkB suggests an anti-angiogenic role for SLURP1 in the transparent cornea. Exp. Eye Res. 164:118-128. PMID: 28803936.
  16. Tiwari, A., Loughner, C.L., Swamynathan, S. and Swamynathan, S.K.* 2017. KLF4 plays an essential role in corneal epithelial homeostasis by promoting epithelial cell fate and suppressing epithelial–mesenchymal transition. Invest. Ophthalmol. Vis. Sci. 58: 2785-2795. PMID: 28549095.
  17. Loughner, C.L., Tiwari, A., Kenchegowda, D., Swamynathan, S. and Swamynathan, S.K.* 2017. Spatiotemporally controlled ablation of Klf5 results in dysregulated epithelial homeostasis in adult mouse corneas. Invest. Ophthalmol. Vis. Sci. 58(11):4683-4693. PMID: 28910443.
  18. Tiwari, A., Swamynathan, S., Alexander, N., Gnalian, J., Tian, S., Kinchington, P.R. and Swamynathan, S.K.* 2019. KLF4 regulates corneal epithelial cell cycle progression by suppressing canonical TGF-b signaling and upregulating CDK inhibitors P16 and P27. Invest. Ophthalmol. Vis. Sci. 60(2):731-740. PMID: 30786277.
  19. Swamynathan, S., Tiwari, A., Loughner, C.L., Gnalian, J., Alexander, N., Jhanji, V., Swamynathan, S.K.* 2019. The secreted Ly6/uPAR-related protein-1 suppresses neutrophil binding, chemotaxis, and transmigration through human umbilical vein endothelial cells. Sci. Rep. 11;9(1):5898. doi: 10.1038/s41598-019-42437-x. PMID: 30976100.
  20. Campbell, G., Swamynathan, S., Tiwari, A., and Swamynathan, S.K.* 2019. The secreted Ly-6/uPAR related protein-1 (SLURP1) stabilizes epithelial cell junctions and suppresses TNF-α-induced cytokine production. Biochem Biophys Res Commun. 517(4):729-734. PMID: 31387745.
  21. Tiwari, A., Swamynathan, S., Jhanji, V. and Swamynathan, S.K.* 2020. KLF4 coordinates corneal epithelial apical-basal polarity and plane of cell division, and is downregulated in ocular surface squamous neoplasia. Invest. Ophthalmol. Vis. Sci. 61(5):15. doi: 10.1167/iovs.61.5.15. PMID: 32396634.
  22. Tiwari, A., Swamynathan, S., Campbell, G., Jhanji, V., and Swamynathan, S.K.* 2020. BMP6 regulates corneal epithelial cell stratification by coordinating their proliferation and differentiation and Is upregulated in pterygium. Invest Ophthalmol Vis Sci. 61(10):46. doi: 10.1167/iovs.61.10.46. PMID:32845956
  23. Swamynathan, S., Campbell. G., Tiwari, A., and Swamynathan, S.K.* 2022. Secreted Ly-6/uPAR-related protein-1 (SLURP1) is a pro-differentiation factor that stalls G1-S transition during corneal epithelial cell cycle progression. Ocul Surf. 24:1-11. doi: 10.1016/j.jtos.2021.12.006. PMID: 34923162
  24. Swamynathan, S., Campbell. G., Sohnen, P., Kaur, S., St. Leger, A. J. and Swamynathan, S.K.* 2024. The secreted Ly6/uPAR-related protein 1 (Slurp1) modulates corneal angiogenic inflammation via NF-kB signaling. Invest Ophthalmol. Vis. Sci. 65(1):37. doi: 10.1167/iovs.65.1.37. PMID: 38252525.
  25. Wells, A., Wang, Y., Shao, H., Sohnen, P., and Swamynathan, S.K. 2024. A CXCR3-Activating Peptide Increases Tear Break Up Time and Corrects Corneal haze in a Rabbit Model of Environmental Dry Eye. J. Pharmacy Pharmacol. Res. 8 (2): 12-19.