Effects of keratin6/16 heterodimer on diabetic wound healing treatment with topical metformin

Abstract

Diabetes is an important public health problem, and it is well known that healing processes are impaired in diabetic wounds as one of its complications. Keratins are structural proteins found in skin cells and play a vital role in wound healing and skin integrity. While there is increasing interest in the anti-inflammatory properties of metformin, a drug commonly used for diabetes, its potential effect on wound healing and keratins is not yet fully understood. In this context, it was aimed to evaluate how metformin administration affects keratin 6 and keratin 16 expression at both mRNA and protein levels. In this study conducted on diabetic rats, the effects of topically applied metformin on keratins in wound healing were investigated. Then, protein and mRNA expression levels of keratin 6 and keratin 16 in treated wounds were compared with untreated wounds using reverse transcription polymerase chain reaction and immunohistochemistry methods. The results of the study are likely to detail changes in the expression levels of keratin 6 and keratin 16 after metformin administration. This information will shed light on how metformin affects the molecular mechanisms involved in wound healing, particularly concerning these important structural proteins. Understanding these changes may provide insight into potential therapeutic approaches to improve diabetic wound healing. By elucidating the effect of metformin on keratin expression, the study may contribute to the development of targeted therapies aimed at improving the healing process in diabetic wounds.

Keywords

• Diabetes mellitus
• diabetic wound model
• keratin
• metformin
• K6/16

References

1. Ahmed, R. R., Mahmoud, A., Ahmed, O. M., Metwalli, A., & Ebaid, H. (2015). Up-regulation of Hsp72 and keratin16 mediates wound healing in streptozotocin diabetic rats. Biological Research, 48, 54.
2. Andrade, T. A. M., Masson-Meyers, D. S., Caetano, G. F., Terra, V. A., Ovidio, P. P., Jordão-Júnior, A. A., & Frade, M. A. C. (2017). Skin changes in streptozotocin-induced diabetic rats. Biochemical and Biophysical Research Communications, 490(4), 1154-1161.
3. Baek, E. J., Jung, D. Y., Seung, N. R., Jang, Y. J., Park, E. J., & Kim, K. H. (2024). Immunohistochemical differentiation of keratins and involucrin between palmar psoriasis, chronic hand eczema and hyperkeratotic hand eczema. Contact Dermatitis, 90(4).
4. Bellavia, G., Fasanaro, P., Melchionna, R., Capogrossi, M. C., & Napolitano, M. (2014). Transcriptional control of skin reepithelialization. Journal of Dermatological Science, 73(1), 3-9.
5. Blakytny, R., & Jude, E. B. (2009). Altered molecular mechanisms of diabetic foot ulcers. The International Journal of Lower Extremity Wounds, 8(2), 95-104.
6. Blumer, S., Khan, P., Artysh, N., Plappert, L., Savic, S., Knudsen, L., ... & Hostettler, K. E. (2024). The use of cultured human alveolar basal cells to mimic honeycomb formation in idiopathic pulmonary fibrosis. Respiratory Research, 25(1), 26.
7. Cheng, J. R., Mao, H., Hui, H. Z., Li, S., Wan, Y. F., & Shi, B. J. (2024). A recurrent missense mutation in the KRT16 gene causing pachyonychia congenita in a patient. International Journal of Dermatology, 63(2), e47-e49.
8. Chu, H. T., Dinh Duong, T. A., Le, D. H., Le, T. V., Nguyen, B. B., Dang, C. V., & Vu, Q. V. (2023). Phenotype and genotype features of Vietnamese children with pachyonychia congenita. Pediatrics and Neonatology, 64(4), 405-410.

9. Cohen, E., Johnson, C. N., Wasikowski, R., Billi, A. C., Tsoi, L. C., Kahlenberg, J. M., ... & Coulombe, P. A. (2024). Significance of stress keratin expression in normal and diseased epithelia. iScience, 27(2), 108805.
10. Franke, W. W., Schiller, D. L., Moll, R., Winter, S., Schmid, E., Engelbrecht, I., ... & Platzer, B. (1981). Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues. Journal of Molecular Biology, 153(4), 933-959.
11. Freedberg, I. M., Tomic-Canic, M., Komine, M., & Blumenberg, M. (2001). Keratins and the keratinocyte activation cycle. The Journal of Investigative Dermatology, 116(5), 633-640.
12. Ghatak, S., Hemann, C., Boslett, J., Singh, K., Sharma, A., El Masry, M. S., ... & Sen, C. K. (2023). Bacterial pyocyanin ınducible keratin 6A accelerates closure of epithelial defect under conditions of mitochondrial dysfunction. Journal of Investigative Dermatology, 143(10), 2052-2064.
13. Graham, G. G., Punt, J., Arora, M., Day, R. O., Doogue, M. P., Duong, J. K., ... & Williams, K. M. (2011). Clinical pharmacokinetics of metformin. Clinical Pharmacokinetics, 50(2), 81-98.
14. Gravino, M., Locci, F., Tundo, S., Cervone, F., Savatin, D. V., & De Lorenzo, G. (2017). Immune responses induced by oligogalacturonides are differentially affected by AvrPto and loss of BAK1/BKK1 and PEPR1/PEPR2. Molecular Plant Pathology, 18(4), 582–595.
15. Groh, N., & Magin, T. M. (2023). Pseudomonas-derived pyocyanin links oxidative stress and keratin 6 expression to wound healing. The Journal of Investigative Dermatology, 143(10), 1865-1867.
16. Hatzfeld, M., & Weber, K. (1990). The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression. The Journal of Cell Biology, 110(4), 1199-1210.
17. Herrmann, H., Strelkov, S. V., Burkhard, P., & Aebi, U. (2009). Intermediate filaments: primary determinants of cell architecture and plasticity. The Journal of Clinical Investigation, 119(7), 1772-1783.
18. Hobbs, R. P., Lessard, J. C., & Coulombe, P. A. (2012). Keratin intermediate filament proteins - novel regulators of inflammation and immunity in skin. Journal of Cell Science, 125(Pt 22), 5257-5258.
19. Howlett, H. C. S., & Bailey, C. J. (2007). Galegine and antidiabetic plants. In: Bailey C. J., Campbell I. W., Chan J. C. N., Davidson J. A., Howlett H. C. S., Ritz P. (eds) Metformin - The Gold Standard: A Scientific Handbook (pp. 3-9). Wiley, Chichester.
20. Jacob, J. T., Coulombe, P. A., Kwan, R., & Omary, M. B. (2018). Types I and II keratin intermediate filaments. Cold Spring Harbor Perspectives in Biology, 10(4), a018275.
21. Karimipour, D. J., Rittié, L., Hammerberg, C., Min, V. K., Voorhees, J. J., Orringer, J. S., ... & Fisher, G. J. (2009). Molecular analysis of aggressive microdermabrasion in photoaged skin. Archives of Dermatology, 145(10), 1114-1122.
22. Khanra, R., Dewanjee, S., K Dua, T., Sahu, R., Gangopadhyay, M., De Feo, V., & Zia-Ul-Haq, M. (2015). Abroma augusta L. (Malvaceae) leaf extract attenuates diabetes induced nephropathy and cardiomyopathy via inhibition of oxidative stress and inflammatory response. Journal of Translational Medicine, 13, 6.
23. Kim, J. H., Yoon, N. Y., Kim, D. H., Jung, M., Jun, M., Park, H. Y., ... & Choi, E. H. (2018). Impaired permeability and antimicrobial barriers in type 2 diabetes skin are linked to increased serum levels of advanced glycation end-product. Experimental Dermatology, 27(8), 815-823.
24. Koch, P. J., & Roop, D. R. (2004). The role of keratins in epidermal development and homeostasis--going beyond the obvious. The Journal of Investigative Dermatology, 123(5), x-xi.
25. Krzyszczyk, P., Schloss, R., Palmer, A., & Berthiaume, F. (2018). The role of macrophages in acute and chronic wound healing and interventions to promote pro-wound healing phenotypes. Frontiers in Physiology, 9, 419. Lima, A. L., Illing, T., Schliemann, S., & Elsner, P. (2017). Cutaneous manifestations of diabetes mellitus: A review. American Journal of Clinical Dermatology, 18(4), 541-553.
26. Majid, O. W. (2024). Preliminary evidence of impaired oral wound healing in e-cigarette users: a call for perioperative vaping cessation. Evidence-Based Dentistry, 1-2.
27. Mansbridge, J. N., & Knapp, A. M. (1987). Changes in keratinocyte maturation during wound healing. The Journal of Investigative Dermatology, 89(3), 253-263.
28. Martin P. (1997). Wound healing--aiming for perfect skin regeneration. Science, 276(5309), 75-81.
29. Mayet, N., Choonara, Y. E., Kumar, P., Tomar, L. K., Tyagi, C., Du Toit, L. C., & Pillay, V. (2014). A comprehensive review of advanced biopolymeric wound healing systems. Journal of Pharmaceutical Sciences, 103(8), 2211-2230.
30. McGowan, K. M., & Coulombe, P. A. (1998). Onset of keratin 17 expression coincides with the definition of major epithelial lineages during skin development. The Journal of Cell Biology, 143(2), 469-486.
31. Michalak‐Micka, K., Tenini, C., Böttcher‐Haberzeth, S., Mazzone, L., Pontiggia, L., Klar, A. S., ... & Biedermann, T. (2024). The expression pattern of cytokeratin 6a in epithelial cells of different origin in dermo‐epidermal skin substitutes in vivo. Biotechnology Journal, 19(1), 2300246.
32. Moll, R., Franke, W. W., Schiller, D. L., Geiger, B., & Krepler, R. (1982). The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell, 31(1), 11-24.
33. Niu, Y., Li, F., Tang, B., Shi, Y., Hao, Y., & Yu, P. (2014). Clinicopathological correlation and prognostic significance of sonic hedgehog protein overexpression in human gastric cancer. International Journal of Clinical and Experimental Pathology, 7(8), 5144-5153.
34. Oshima R. G. (2002). Apoptosis and keratin intermediate filaments. Cell Death and Differentiation, 9(5), 486-492.
35. Paladini, R. D., Takahashi, K., Bravo, N. S., & Coulombe, P. A. (1996). Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16. The Journal of Cell Biology, 132(3), 381-397.
36. Pan, X., Hobbs, R. P., & Coulombe, P. A. (2013). The expanding significance of keratin intermediate filaments in normal and diseased epithelia. Current Opinion in Cell Biology, 25(1), 47-56.
37. Park, H. Y., Kim, J. H., Jung, M., Chung, C. H., Hasham, R., Park, C. S., & Choi, E. H. (2011). A long-standing hyperglycaemic condition impairs skin barrier by accelerating skin ageing process. Experimental Dermatology, 20(12), 969-974.
38. Pastar, I., Stojadinovic, O., Yin, N. C., Ramirez, H., Nusbaum, A. G., Sawaya, A., ... & Tomic-Canic, M. (2014). Epithelialization in wound healing: A comprehensive review. Advances in Wound Care, 3(7), 445-464.
39. Patel, G. K., Wilson, C. H., Harding, K. G., Finlay, A. Y., & Bowden, P. E. (2006). Numerous keratinocyte subtypes involved in wound re-epithelialization. The Journal of Investigative Dermatology, 126(2), 497–502.
40. Raja, Sivamani, K., Garcia, M. S., & Isseroff, R. R. (2007). Wound re-epithelialization: modulating keratinocyte migration in wound healing. Frontiers in Bioscience: A Journal and Virtual Library, 12, 2849-2868.
41. Ramey-Ward, A. N., Walthall, H. P., Smith, S., & Barrows, T. H. (2023). Human keratin matrices promote wound healing by modulating skin cell expression of cytokines and growth factors. Wound Repair and Regeneration, 1-11.
42. Savatin, D. V., Bisceglia, N. G., Marti, L., Fabbri, C., Cervone, F., & De Lorenzo, G. (2014). The Arabidopsis nucleus-and phragmoplast-localized kinase1-related protein kinases are required for elicitor-induced oxidative burst and immunity. Plant Physiology, 165(3), 1188-1202.
43. Schweizer, J., Bowden, P. E., Coulombe, P. A., Langbein, L., Lane, E. B., Magin, T. M., ... & Wright, M. W. (2006). New consensus nomenclature for mammalian keratins. The Journal of Cell Biology, 174(2), 169-174.
44. Shavandi, A., Silva, T. H., Bekhit, A. A., & Bekhit, A. E. A. (2017). Keratin: dissolution, extraction and biomedical application. Biomaterials Science, 5(9), 1699-1735.
45. Sellappan, L. K., & Manoharan, S. (2024). Fabrication of bioinspired keratin/sodium alginate based biopolymeric mat loaded with herbal drug and green synthesized zinc oxide nanoparticles as a dual drug antimicrobial wound dressing. International Journal of Biological Macromolecules, 259(Pt 1), 129162.
46. Simsek, A., & Ozmen, O. (2024). Histopathological and immunohistochemical effects of tarantula cubensis extract on mucosal healing in rats. Journal of Veterinary Dentistry, 41(1), 17-25.
47. Stojadinovic, O., Brem, H., Vouthounis, C., Lee, B., Fallon, J., Stallcup, M., ... & Tomic-Canic, M. (2005). Molecular pathogenesis of chronic wounds: the role of beta-catenin and c-myc in the inhibition of epithelialization and wound healing. The American Journal of Pathology, 167(1), 59-69.
48. Sun, C., Huang, Y., Wang, L., Deng, J., Qing, R., Ge, X., ... & Hao, S. (2024). Engineered keratin/bFGF hydrogel to promote diabetic wound healing in rats. International Journal of Biological Macromolecules, 261(Pt 1), 129725.
49. Sun, C., Liu, W., Wang, L., Meng, R., Deng, J., Qing, R., ... & Hao, S. (2023). Photopolymerized keratin-PGLa hydrogels for antibiotic resistance reversal and enhancement of infectious wound healing. Materials Today Bio, 23, 100807.
50. Sun, T. T., Eichner, R., Nelson, W. G., Vidrich, A., & Woodcock-Mitchell, J. (1983). Keratin expression during normal epidermal differentiation. Current Problems in Dermatology, 11, 277-291.
51. Takahashi, K., Yan, B., Yamanishi, K., Imamura, S., & Coulombe, P. A. (1998). The two functional keratin 6 genes of mouse are differentially regulated and evolved independently from their human orthologs. Genomics, 53(2), 170-183.
52. Tavakoli, M., Mirhaj, M., Varshosaz, J., Al-Musawi, M. H., Almajidi, Y. Q., Danesh Pajooh, A. M., ... & Esfahani, S. N. (2023). Keratin- and VEGF-incorporated honey-based sponge-nanofiber dressing: An ideal construct for wound healing. ACS Applied Materials & Interfaces, 15(48), 55276-55286.
53. Usui, M. L., Underwood, R. A., Mansbridge, J. N., Muffley, L. A., Carter, W. G., & Olerud, J. E. (2005). Morphological evidence for the role of suprabasal keratinocytes in wound reepithelialization. Wound Repair and Regeneration, 13(5), 468-479.
54. Velnar, T., Bailey, T., & Smrkolj, V. (2009). The wound healing process: an overview of the cellular and molecular mechanisms. The Journal of International Medical Research, 37(5), 1528-1542.
55. Wawersik, M. J., Mazzalupo, S., Nguyen, D., & Coulombe, P. A. (2001). Increased levels of keratin 16 alter epithelialization potential of mouse skin keratinocytes in vivo and ex vivo. Molecular Biology of The Cell, 12(11), 3439-3450.
56. Wawersik, M., & Coulombe, P. A. (2000). Forced expression of keratin 16 alters the adhesion, differentiation, and migration of mouse skin keratinocytes. Molecular Biology of The Cell, 11(10), 3315-3327.
57. Wikramanayake, T. C., Stojadinovic, O., & Tomic-Canic, M. (2014). Epidermal differentiation in barrier maintenance and wound healing. Advances in Wound Care, 3(3), 272-280.
58. Winkfield, K. M., Hughes, R. T., Brown, D. R., Clohessy, R. M., Holder, R. C., ... & Burnett, L. R. (2024). Randomized pilot study of a keratin-based topical cream for radiation dermatitis in breast cancer patients. Technology in Cancer Research & Treatment, 23, 15330338231222137.
59. Wojcik, S. M., Bundman, D. S., & Roop, D. R. (2000). Delayed wound healing in keratin 6a knockout mice. Molecular and Cellular Biology, 20(14), 5248-5255.
60. Wong, P., & Coulombe, P. A. (2003). Loss of keratin 6 (K6) proteins reveals a function for intermediate filaments during wound repair. The Journal of Cell Biology, 163(2), 327-337.
61. Zhang, L. J. (2018). Keratins in skin epidermal development and diseases. In: Blumenberg, M., Surguchov, A. (eds) Keratin (pp. 1-17). Intech Open London, UK.