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

成纤维细胞(FBS)是各种组织合成和维持细胞外基质(ECM)动态平衡的主要细胞。FBS对外界刺激做出反应，并相应地调整其功能(增殖、迁移、激活)。过量的FB激活会导致ECM(瘢痕/纤维化)的过度产生，而反之则会导致结构的不稳定。去整合素和金属蛋白酶-15(ADAM15)和ADAM17是FBS中两个高表达的ADAM，被错误地认为具有相似的功能，尽管最近的发现表明情况并非如此。我们先前在心血管疾病模型中的研究表明，迫切需要了解每个ADAM独特的机械功能，以及它们调节FB功能和ECM重塑的基本过程。ADAM15是唯一一个切割ECM蛋白的ADAM，而ADAM17是具有&gt；80底物的功能最广泛的脱落酶。ADAMS具有处理膜结合蛋白并与整合素相互作用(通过其去整合素结构域)的能力，但尚不确定哪种机制优先，以及这是否与ADAM或刺激的性质有关。我们推测，在FBS中，ADAM15主要通过其C末端介导的信号以及与整合素相互作用和激活相关的细胞内信号而发挥作用；而ADAM17的功能主要是通过蛋白水解性脱落膜结合蛋白(生长因子、细胞因子)，提高其生物利用度及其下游途径。在接下来的5年里，我们将研究ADAM15和ADAM17在药物刺激和机械刺激下FBS在ECM产生和动态平衡中的作用的比较。我们将使用主要的FB培养系统，并将使用siRNA(打乱的siRNA将用作平行对照)敲除ADAM15或ADAM17。随后，FBS将接受药物刺激(血管紧张素II或苯肾上腺素)或机械应激(有节奏或静态拉伸，FlexCell5000系统)的治疗。FB的活性(ECM蛋白的合成、收缩)、增殖、迁移和存活将在对照组、ADAM15和ADAM17缺陷的FBS(±刺激)中进行研究。将进行详细的分子和机制分析，以比较和对比这两个ADAMS在激动剂诱导的FBS和机械激活的FBS以及整个ECM重塑中的作用。我的实验室在研究ECM重塑和FB功能方面有丰富的经验。我们拥有进行上述实验所需的所有设备和专业知识。这些实验将揭示ADAM15和-17在FB功能和ECM重塑中的新作用机制，并通过确定这些ADAM15和-17在FBS中的蛋白分解和机械感觉功能，将在细胞生理学和自然科学中创造新的知识，这将有助于细胞和机械生物学领域的研究。

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.