Genetic and pharmacological tools to study the role of discoidin domain receptors in kidney disease.

Following injury the kidney undergoes a repair process, which results in replacement of the injured tissue with little evidence of damage. However, repetitive injuries or inability of the kidney to stop the repair process result in abnormal deposition of extracellular matrix (ECM) components leading to fibrosis and organ dysfunction. The synthesis/degradation of ECM components is finely regulated by several factors, including discoidin domain receptors (DDRs). These are receptor tyrosine kinases that are activated by collagens. Upon activation, DDRs control several cell functions that, when exacerbated, contribute to kidney injury and fibrosis. DDRs are undetectable in healthy kidney, but become rapidly upregulated in several kidney fibrotic conditions, thus making them attractive anti-fibrotic targets. DDRs contribute to kidney injury and fibrosis by promoting apoptosis of injured kidney cells, stimulating the production of pro-inflammatory cytokines, and regulating the production of ECM components. They achieve these effects by activating canonical intracellular molecules or by directly interacting with nuclear chromatin and promoting the transcription of pro-fibrotic genes. The goal of this review is to highlight canonical and non-canonical mechanisms whereby DDRs contribute to kidney injury/fibrosis. This review will summarize key findings obtained using cells and mice lacking DDRs and it will discuss the discovery and development of targeted DDR small molecule- and antisense-based inhibitors. Understanding the molecular mechanisms whereby DDRs control kidney injury and fibrosis might enable us to not only develop more selective and potent inhibitors, but to also determine when DDR inhibition needs to be achieved to prevent and/or halt the development of kidney fibrosis.

The Collagen Receptor Discoidin Domain Receptor 1b Enhances Integrin ß1-Mediated Cell Migration by Interacting With Talin and Promoting Rac1 Activation.

Integrins and discoidin domain receptors (DDRs) 1 and 2 promote cell adhesion and migration on both fibrillar and non fibrillar collagens. Collagen I contains DDR and integrin selective binding motifs; however, the relative contribution of these two receptors in regulating cell migration is unclear. DDR1 has five isoforms (DDR1a-e), with most cells expressing the DDR1a and DDR1b isoforms. We show that human embryonic kidney 293 cells expressing DDR1b migrate more than DDR1a expressing cells on DDR selective substrata as well as on collagen I in vitro. In addition, DDR1b expressing cells show increased lung colonization after tail vein injection in nude mice. DDR1a and DDR1b differ from each other by an extra 37 amino acids in the DDR1b cytoplasmic domain. Interestingly, these 37 amino acids contain an NPxY motif which is a central control module within the cytoplasmic domain of ß integrins and acts by binding scaffold proteins, including talin. Using purified recombinant DDR1 cytoplasmic tail proteins, we show that DDR1b directly binds talin with higher affinity than DDR1a. In cells, DDR1b, but not DDR1a, colocalizes with talin and integrin ß1 to focal adhesions and enhances integrin ß1-mediated cell migration. Moreover, we show that DDR1b promotes cell migration by enhancing Rac1 activation. Mechanistically DDR1b interacts with the GTPase-activating protein (GAP) Breakpoint cluster region protein (BCR) thus reducing its GAP activity and enhancing Rac activation. Our study identifies DDR1b as a major driver of cell migration and talin and BCR as key players in the interplay between integrins and DDR1b in regulating cell migration.

DDR1 contributes to kidney inflammation and fibrosis by promoting the phosphorylation of BCR and STAT3.

Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase activated by collagen, contributes to chronic kidney disease. However, its role in acute kidney injury and subsequent development of kidney fibrosis is not clear. Thus, we performed a model of severe ischemia/reperfusion-induced acute kidney injury that progressed to kidney fibrosis in WT and Ddr1-null mice. We showed that Ddr1-null mice had reduced acute tubular injury, inflammation, and tubulointerstitial fibrosis with overall decreased renal monocyte chemoattractant protein (MCP-1) levels and STAT3 activation. We identified breakpoint cluster region (BCR) protein as a phosphorylated target of DDR1 that controls MCP-1 production in renal proximal tubule epithelial cells. DDR1-induced BCR phosphorylation or BCR downregulation increased MCP-1 secretion, suggesting that BCR negatively regulates the levels of MCP-1. Mechanistically, phosphorylation or downregulation of BCR increased ß-catenin activity and in turn MCP-1 production. Finally, we showed that DDR1-mediated STAT3 activation was required to stimulate the secretion of TGF-ß. Thus, DDR1 contributes to acute and chronic kidney injury by regulating BCR and STAT3 phosphorylation and in turn the production of MCP-1 and TGF-ß. These findings identify DDR1 an attractive therapeutic target for ameliorating both proinflammatory and profibrotic signaling in kidney disease.

Inhibitory effects of recombinant RTS-jerdostatin on integrin α1ß1 function during adhesion, migration and proliferation of rat aortic smooth muscle cells and angiogenesis.

Jerdostatin, a short RTS-disintegrin cloned from venom gland mRNA of Protobothrops jerdonii, selectively blocks the adhesion of α1ß1 integrin to collagen IV. Integrin α1ß1 is highly expressed in smooth muscle cells (SMC) surrounding small blood vessels and vascular endothelial cells. Vascular SMC adhesion, migration and proliferation are important processes during normal vascular development. Using recombinant jerdostatin we have investigated the role of the α1ß1 integrin on the adhesion of vascular SMC to collagen IV, and the potential relevance of blocking this crucial component of focal adhesions as an anti-angiogenic strategy. Our results show that jerdostatin does not interact with canonical collagen-binding site on the isolated A-domain of the α1 integrin subunit. r-Jerdostatin inhibited the adhesion of RASMCs to immobilized CB3 fragment in a dose-dependent manner, triggering to round-up, retraction, and finally detachment of the cells. r-Jerdostatin did not affect the adhesion of human SMCs to CB3, presumably because the high expression of α2ß1 integrin compensated for α1ß1 integrin blockage by jerdostatin. r-Jerdostatin dose-dependently inhibited α1ß1 integrin-dependent HUVEC tube formation. However, VEGF-driven tube formation in the matrigel assay was only completely abolished when binding of integrin α2ß1 to collagen was also inhibited by the C-type lectin-like rhodocetin. As a whole, our work emphasizes the relevance of using specific inhibitors for dissecting the role of α1ß1 integrin in physiological and pathological conditions.

Recombinant expression of mutants of the Frankenstein disintegrin, RTS-ocellatusin. Evidence for the independent origin of RGD and KTS/RTS disintegrins.

The requirements to transform a short disintegrin of the RGD clade into an RTS disintegrin, were investigated through the generation of recombinant mutants of ocellatusin in which the RGD tripeptide was substituted for RTS in different positions along the integrin-specificity loop. Any attempt to create an active integrin α(1)ß(1) inhibitory motif within the specificity loop of ocellatusin was unsuccessful. Replacing the whole RGD-loop of ocellatusin by the RTS-loop of jerdostatin was neither sufficient for confering α(1)ß(1) binding specificity to this ocellatusin-RTS Frankenstein(2) mutant. Factors other than the integrin-binding loop sequence per se are thus required to transform a disintegrin scaffold from the RGD clade into another scaffold from the RTS/KTS clade. Moreover, our results provide evidences, that the RTS/KTS short disintegrins have potentially been recruited into the venom gland of Eurasian vipers independently from the canonical neofunctionalization pathway of the RGD disintegrins. PCR-amplifications of jerdostatin-like sequences from a number of taxa across reptiles, including snakes (Crotalinae, Viperinae, and Elapidae taxa) and lizards (Lacertidae and Iguanidae) clearly showed that genes coding for RTS/KTS disintegrins existed long before the split of Lacertidae and Iguania, thus predating the recruitment of the SVMP precursors of disintegrins, providing strong support for the view of an independent evolutionary history of the RTS/KTS and the RGD clades of short disintegrins.

Recombinant expression in human cells of active integrin alpha 1 beta 1-blocking RTS-disintegrin jerdostatin.

Jerdostatin, an RTS short disintegrin cloned from Protobothrops jerdonii and recombinantly produced in Escherichia coli, is a potent and specific antagonist of the alpha(1)beta(1) integrin. Jerdostatin selectively blocked the adhesion of alpha(1)beta(1)-K562 cell to collagens I and IV in vitro and angiogenesis in vivo. Here we report the recombinant production of jerdostatin in a mammalian cell system, a prerequisite for developing a conditional transgenic mouse to investigate the effect of systemic expression of jerdostatin on tumor development. For proper export of jerdostatin, a secretion leader sequence was engineered at the protein's N-terminus. A FLAG epitope was also included at the N-terminus of the mature disintegrin to facilitate its isolation and characterization of recombinant jerdostatin (rJerd). This pRc-CMV/FLAG-rJerd construct was transiently expressed in HEK-293 cells and was efficiently secreted into the culture medium. rJerd bound to recombinant soluble alpha(1)beta(1) integrin in a saturable and cation-independent manner. Soluble rJerd also inhibited the binding of alpha(1)beta(1) integrin to the CB3 fragment of collagen IV in a dose-dependent manner (IC(50) 570 nM). Mammalian cell-expressed jerdostatin disrupted the adhesion of RuGli cells to collagen IV. Our results highlight pRc-CMV/FLAG-rJerd as a suitable construct for expressing soluble active alpha(1)beta(1)-blocking jerdostatin in a mammalian cell system.