Kidney Injury-Repair: The kidney has the capacity to regenerate completely after various acute insults. Regeneration by surviving tubular epithelial cells that proliferate and migrate to reepithelialize the tubulus is the predominant mechanism of repair. Several metalloprotease cleavage-driven events (ectodomain shedding) are implicated in the underlying processes of renal regeneration. For example, in kidney proximal tubular cells, the cleavage activated epidermal growth factor receptor (EGFR) ligands EGF, heparin binding-EGF (HB-EGF), amphiregulin (AR) and epiregulin (ER) can induce regenerative proliferation, cell motility and migration. In vivo, functional EGFR is required for recovery after renal injury and its activation enhances regeneration.
Maladaptive Repair (Fibrosis): Severe or chronic kidney injury, in contrast, leads to sustained activation of the EGFR, which is pro-fibrotic, and leads to scaring of the kidney (fibrosis) and loss of function (Kidney failure). It is not known which specific EGF ligands are involved in this process and why sustained EGFR activation is pro-fibrotic.
Organ Cross talk: AKI is clinically associated with organ dysfunction in the lung, heart, liver and brain (among others). These effects are NOT simply explained by uremia, electrolyte imbalances or changes in the amount of body fluid. Instead, there is evidence that AKI and CKD cause inflammation and organd dysfunction in distant organs. This is in part mediated by soluble factors released from the kidney and potentially other secondary sources in the body.
We study this process in detail in mouse models of kidney injury or fibrosis and in tubular cells in culture.
Ectodomain shedding: Only two a-disintegrin-and-metalloproteases, ADAM10 and 17, cleave most physiologically and disease-relevant substrates (>100). A process termed ectodomain cleavage or ectodomain shedding. This explains why metalloprotease inhibitors have failed clinically in part due to negative side effects induced by broadly inhibited substrate cleavage. Regulated substrates include e.g. the precursors of growth factors like epidermal growth factor (EGF) ligands, cytokines (TNFα), as well as adhesion molecules (e.g. CD44, ICAM-1). Interestingly, also many of the involved growth factor or cytokine receptors undergo regulated shedding, allowing the possibility of negative regulation on the receptor level or the production of decoy receptors. After ectodomain shedding, the subsequent release by γ-secretase of the intracellular-domains from proteins such as Notch, NRG1, Erb4 or CD44, which regulate transcriptional activity in the nucleus, provides further levels of complexity. Ectodomain shedding therefore affects numerous cellular processes, including for example growth, adhesion and motility of cells, throughout life.
A complex interplay between cellular signaling pathways and the shedding of bioactive molecules determines the ultimately expressed phenotype of the cell. The broad array of substrates and biological phenotypes regulated, illustrates the need for tight control. Unfortunately it is largely unknown, how ectodomain cleavage is regulated and made substrate-specific. Furthermore, extensive cross-talk and complexity among signaling networks, proteases, and their substrates make understanding sheddase regulation on a component-by-component basis challenging (2). Therefore, in order to develop improved therapeutics, a strong need exists to understand how the balance of sheddase-mediated proteolysis integrates multiple layers of signaling networks to coordinately influence cell behavior in various disease contexts.
To meet these challenges, we take a systemic view in our approach to investigate cleavage regulation.
Our research aims to delineate the signaling networks and molecular mechanisms that determine substrate specificity of cleavage by combining
(1) in vitro and in vivo approaches aimed at identifying the regulated cleavage mechanism and its physiological significance in disease models, with
(2) integrated biocomputational network analysis of a large dataset relevant to cleavage regulation.
This approach aims at identifying and validating specific signaling nodes that regulate the cleavage of specific substrates and relevant phenotypes. We generated this extensive and still growing dataset with a large scale shRNA screen targeting all human kinases and phosphatases, testing their effect on the induced cleavage of the EGF ligand TGFα. A core set of candidate regulators (activators and inhibitors), some of which regulate cleavage on the substrate level without affecting metalloprotease activity (Dang et al. PNAS 2013), was then further analyzed with biocomputational methods and in vitro validation (Wilson et al. Mol Cancer Research 2018). This dataset and analysis is enriched by our findings on specific cleavage mechanisms of substrates (e.g. Dang et al PNAS 2013, Hartmann et al. Mol Cancer Research 2015; Hartmann et al. JBC 2015).