Deciphering the role of ADAM15 vs. ADAM17 in fibroblast function: Mechanobiology vs. Proteolysis

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Fibroblasts (FBs) are the main cells for synthesis and homeostasis of the extracellular matrix (ECM) in various tissues. FBs respond to external stimuli and adjust their function (proliferation, migration, activation) accordingly. Excess FB activation leads to overproduction of ECM (scar/fibrosis), while the inverse results in structural instability. Disintegrin and metalloproteinase-15 (ADAM15) and ADAM17 are two highly expressed ADAMs in FBs, and are (incorrectly) assumed to have similar functions, though recent findings suggest otherwise. Our prior research in cardiovascular disease models has revealed the dire need to understand the unique mechanistic function of each ADAM, and the fundamental processes by which they regulate FB function and ECM remodeling. ADAM15 is unique as the only ADAM to cleave an ECM protein, whereas ADAM17 is the most versatile sheddase with >80 substrates. ADAMs have the ability to proteolytically process membrane-bound proteins and to also interact with integrins (through their disintegrin domain), but it is yet to be determined which mechanism takes priority, and if this is specific to an ADAM or to the nature of the stimulus. We hypothesize that in FBs, ADAM15 predominantly functions through its C-terminus-mediated signaling, and by interacting with integrins and activating the associated intracellular signaling; whereas the function of ADAM17 is primarily through proteolytic shedding of membrane-bound proteins (growth factors, cytokines) enhancing their bioavailability and their downstream pathways. Over the next 5 years, we will investigate the comparative function of ADAM15 and ADAM17 in the function of FBs in ECM production and homeostasis in response to pharmacological versus mechanical stimuli. We will use a primary FB culture system, and will knockdown ADAM15 or ADAM17 using siRNA (scrambled siRNA will be used as control in parallel). Subsequently, FBs will be treated with pharmacological stimuli (Angiotensin II or Phenylephrine) or subjected to mechanical stress (rhythmic or static stretching, FlexCell5000 system). FB activity (synthesis of ECM proteins, contractility), proliferation, migration, and survival will be investigated in control, ADAM15- and ADAM17-deficient FBs (±stimulus). Detailed molecular and mechanistic analyses will be performed to compare and contrast the role of these two ADAMs in agonist-induced versus mechanical activation of FBs and the overall ECM remodeling. My lab has extensive experience in studying ECM remodeling and FB functions. We have all the equipment and expertise necessary to conduct the described experiments. These experiments will reveal novel mechanism of action for ADAM15 and -17 in FB function and ECM remodeling, and will create new knowledge in cell physiology and natural sciences by determining the proteolytic and mechano-sensory functions of these ADAMs in FBs, which would be beneficial in the fields of cell- and mechano-biology.