Cell surface proteolysis in development, tissue repair, and malignancy

National Institute Of Dental & Craniofacial Research

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Abstract

General Mission Statement: The overall aim of this project is to understand the biochemistry, biology, and pathology of cell surface-associated proteolysis, with an emphasis on defining the contribution of proteolytic pathways to the development, homeostasis, regeneration, and pathology of oral tissues. Ongoing efforts and future plans: A critical role of TMPRSS11 proteases in oral fungal immunity We have previously identified a vertebrate multi-gene cluster encompassing five to seven-closely related membrane-anchored serine proteases (TMPRSS11A-G), encoded by the TMPRSS11A-G genes. In collaboration with Niki Moutsopoulos, we found that members of this protease family, of hitherto unknown function, are particularly highly expressed in oral epithelial cells, as documented by bulk RNA and single cell RNA sequencing of human and mouse tissues. To explore the functions of TMPRSS11 proteases in epithelial biology, we also previously generated a congenic mouse strain with null mutations in all seven Tmprss11 genes. Surprisingly, mice lacking all TMPRSS11 proteases (hereafter Tmprss11-all) were outwardly healthy and fertile. In collaboration with Michail Lionakis, Fungal Pathogenesis Section, NIAID, we explored the role of TMPRSS11 proteases in fungal immunity by oral inoculation of Tmprss11-all mice and wildtype littermates with Candida albicans. Interestingly, Tmprss11-all mice displayed a remarkably increased susceptibility to oral candidiasis, presenting with a 10 to 100-fold increase in fungal colonization of the tongue, buccal mucosa, and palate. This failure to clear the infection was accompanied by the development of epithelial ulcers with neutrophil-dominated immune infiltration. Possibly related to increased fungal susceptibility, toluidine blue dye penetration assays of unchallenged Tmprss11-all mice revealed a compromised pericellular barrier function, and oral tissues presenting histologically with compensatory hyperkeratosis and acanthosis. Because Tmprss11a, Tmprss11b, and Tmprss11d combined give rise to more than 90% of all Tmprss11 mRNA transcripts of the mouse tongue, we next inoculated Tmprss11a, Tmprss11b, and Tmprss11d single-deficient mice with candida albicans. This revealed that mice individually deficient in the three TMPRSS11 proteases displayed normal fungal immunity, suggesting that TMPRSS11 proteases may have redundant functions in oral epithelial biology. We currently are performing additional studies of oral epithelial function as well as a detailed immunophenotyping of Tmprss11-all mice to attempt to elucidate the mechanistic underpinning of the critical role of TMPRSS11 proteases in fungal immunity. A critical role of bone marrow fibrin deposition in chemotherapy-induced myelosuppression Fibrin is a provisional ECM protein that is formed by the polymerization of thrombin-cleaved fibrinogen, followed by cross-linking of the fibrin polymer by coagulation factor XIII. Fibrin is deposited into the extravascular space in response to tissue injury, where it serves to stem the loss of blood, immobilize bacteria, and provide a provisional matrix for tissue regeneration. AA Myelosuppression, leading to life-threatening anemia, is a frequent complication associated with cancer chemotherapy. Myelosuppression is particularly well-documented for 5-Fluorouracil (5- FU), one of the most frequently used cancer chemotherapy drugs. Because persistent fibrin deposition impairs tissue regeneration in a myriad of contexts, we characterized the deposition of fibrin in bone marrow of 5-FU-treated mice. We found that administration of a therapeutic dose of 5-FU to mice leads to pervasive bone marrow fibrin deposition at day three after treatment- initiation, which peaks at day 8, and is resolved by day 15, associated with hematopoietic recovery. Interestingly, mice deficient in the principal fibrinolytic protease zymogen, plasminogen, typically became moribund starting day 8 after 5-FU administration. Importantly, genetic imposition of complete fibrinogen deficiency restored the recovery of plasminogen-deficient mice after 5-FU treatment. To explore the therapeutic potential of modulating fibrinogen expression to support hematopoietic recovery after 5-FU administration, in a collaboration with Dr. Christian Kastrup, Department of Surgery, Medical College of Wisconsin, Wauwatosa, Wisconsin, USA, we targeted fibrinogen expression by systemic administration of liver-targeted siRNA liposomes. Importantly, 5-FU-treated plasminogen-deficient mice displayed dramatically prolonged survival following administration of liver-targeted siRNA liposomes. We are currently exploring the specific mechanism by which bone marrow fibrin deposition suppresses hematopoietic recovery. In this regard, preliminary studies suggest that plasminogen-deficient mice carrying a mutant fibrinogen (fibrinogen-y390-396A) that does not engage the myeloid-specific alphaMbeta2 integrin display an increased tolerance to 5-FU approaching that of fibrinogen-deficient mice. This implies that bone marrow fibrin deposition impairs hematopoietic recovery by recruiting and/or activating one or more myeloid cell populations, and that currently emerging drugs that uncouple fibrin’s hemostatic and inflammatory functions may accelerate hematopoietic recovery after chemotherapy- or radiation- induced myelosuppression.