Project Grant R00HL168365

P16INK4A+ FIBROBLASTS REGULATE EPITHELIAL REGENERATION AFTER INJURY IN LUNG ALVEOLI THROUGH THE SASP - PROJECT SUMMARY ABSTRACT CELLULAR SENESCENCE IS AS AN ACQUIRED STATE CELLS ENTER IN RESPONSE TO ENVIRONMENTAL STRESSORS OR TUMOR EVASION. SENESCENT CELLS CEASE THEIR PROLIFERATIVE CAPACITY AND ENHANCE THEIR ABILITY TO RESPOND AND REGULATE THE MICROENVIRONMENT THROUGH THE SENESCENCE-ASSOCIATED SECRETORY PHONOTYPE (SASP). ANOTHER SHARED CHARACTERISTIC OF CELLULAR SENESCENCE IS THE UPREGULATION OF THE TUMOR SUPPRESSOR CYCLIN INHIBITOR P16INK4A. WITH AGE THERE'S ACCUMULATION OF SENESCENCE CELLS IN TISSUES ALONG WITH THE UPREGULATION OF P16INK4A EXPRESSION. WHILE REMOVAL OF P16INK4A EXPRESSING CELLS USING GENETIC MOUSE TOOLS SLOWS DOWN AGING, IT ALSO ADVERSELY IMPACTS WOUND HEALING DURING INJURY REPAIR, SUGGESTING CONTRADICTORY ROLES OF P16INK4A DURING HOMEOSTASIS AND INJURY RESPONSE. WE GENERATED AN ULTRA-SENSITIVE P16INK4A REPORTER MOUSE LINE NAME, NAMED INK4A H2B-GFP REPORTER-IN-TANDEM, OR INKBRITE TO FURTHER UNDERSTAND IN VIVO P16INK4+ CELLS. IN INKBRITE ADULT LUNGS, P16INK4+ CELLS ARE PREDOMINANTLY WITHIN IMMUNE AND FIBROBLASTS POPULATIONS. P16INK4+ FIBROBLASTS EXPRESS FEATURES OF SENESCENCE INCLUDING POLYPLOIDY, INCREASE IN CELL SIZE, LOW PROLIFERATION CAPACITY AND ABILITY TO PROMOTE AIRWAY EPITHELIAL CELL GROWTH AFTER INJURY. MY MAIN GOAL IS TO DETERMINE IF THE CAPACITY TO PROMOTE REGENERATION IS RESTRICTED TO AIRWAY P16INK4+ AND EPITHELIUM OR OTHER REGIONALLY SPECIFIC P16INK4 EXPRESSING FIBROBLAST CAN SUPPORT EPITHELIAL GROWTH. WHETHER THE CAPACITY TO PROMOTE EPITHELIAL REGENERATION IS RESTRICTED TO AIRWAY P16INK4+ FIBROBLASTS OR OTHER SPATIALLY DEFINED FIBROBLAST SUBPOPULATIONS SUCH AS ALVEOLAR FIBROBLAST CAN PROMOTE EPITHELIAL GROWTH THROUGH SASP, REMAINS UNKNOWN. TO FILL THIS KNOWLEDGE GAP, I WILL ISOLATE ALVEOLAR P16INK4A+ FIBROBLASTS AND DETERMINE THEIR CAPACITY TO PROMOTE EPITHELIAL GROWTH AFTER ACUTE EPITHELIAL INJURY BY 1) EX VIVO 3D CO-CULTURE ASSAY, 2) IDENTIFY THE TRANSCRIBED SASP THROUGH RNA SEQUENCING, AND 3) IN VIVO USING KNOWN SENOLYTICS DASATINIB AND QUERCETIN (D & Q). OUR P16INK4A INDUCTION AND KNOCKDOWN STUDIES SHOWED THE REQUIREMENT OF P16INK4A FOR EXPRESSION OF KNOWN SASP FACTORS SUCH AS IL6, EREG, CCL8. ANOTHER ASPECT THAT HAS BEEN LARGELY UNDEREXPLORED IN THE IDENTITY AND DEPENDENCE P16INK4 EXPRESSION OF SASP FACTORS IN VIVO. FOR THE R00 PHASE OF MY PROPOSED WORK, I WILL FURTHER EXPLORE HOW THE EXPRESSION OF P16INK4 IS ABLE TO REPROGRAM THE SASP TO SUPPORT TISSUE REPAIR, SPECIFICALLY EPITHELIAL REGENERATION. I WILL WITH OUR TOOLS TO FUNCTIONALLY INDUCE AND REMOVE P16INK4 IN FIBROBLASTS AND 1) ASSES EPITHELIAL GROWTH, 2) TRANSCRIPTOME ANALYSIS, AND 3) PROTEOMICS TO CAPTURE SECRETED PROTEINS TO IDENTIFY P16INK4A-DEPENDENT SASP FACTORS. I HAVE EXTENSIVE KNOWLEDGE ON WORKING WITH INKBRITE AND THE TOOLS TO MANIPULATE P16 INK4A EXPRESSION WHICH WILL ALLOW ME TO PURSUE THE PROPOSED WORK WITH EASE. WITH ADDITIONAL TRAINING FROM DRS. PENG AND SHEPPARD I WILL EXPAND MY CURRENT KNOWLEDGE OF LUNG BIOLOGY AND CELLULAR SENESCENCE WHILE ESTABLISHING A NEW PROTOCOL OF IDENTIFYING SECRETED PROTEINS IN VIVO THAT WILL SERVE AS A UNIQUE SKILLSET FOR MY TRANSITION TO MY OWN LAB. THESE STUDIES WILL LAY FOUNDATION FOR OUR UNDERSTANDING OF DIVERSE ROLES OF SASP FACTORS DURING HOMEOSTASIS AND INJURY.