Thrombin stimulates proinflammatory and proliferative responses in primary cultures of human proximal tubule cells

Vesey, David A.; Cheung, Catherine; Kruger, Wade A.; Poronnik, Philip; Gobé, Glenda C.; Johnson, David W.

doi:10.1111/j.1523-1755.2005.00209.x

Cited by 43 publications

(56 citation statements)

References 67 publications

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“…Several models of PAR trans-and coactivation have been proposed (11,(21)(22)(23)(24)(25). Studies showed that platelet-specific PAR1/4 heterodimerization was responsible for thrombininduced platelet aggregation (26).…”

Section: Discussionmentioning

confidence: 99%

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

McLaughlin

Patterson

Malik

2007

Proc. Natl. Acad. Sci. U.S.A.

160

164

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Thrombin activates endothelial cell signaling by cleaving the protease-activated receptor-1 (PAR1). However, the function of the apparently nonsignaling receptor PAR3 also expressed in endothelial cells is unknown. We demonstrate here the crucial role of PAR3 in potentiating the responsiveness of PAR1 to thrombin. We tested the hypothesis that PAR1/PAR3 heterodimerization and its effect in modifying G protein selectivity was responsible for PAR3 regulation of PAR1 sensitivity. Using bioluminescent resonance energy transfer-2, we showed that PAR1 had comparable dimerization affinity for PAR3 as for itself. We observed increased G␣13 coupling between the PAR1/3 heterodimer compared with the PAR1/1 homodimer. Moreover, knockdown of PAR3 moderated the PAR1-activated increase in endothelial permeability. These results demonstrate a role of PAR3 in allosterically regulating PAR1 signaling governing increased endothelial permeability. Because PAR3 is a critical determinant of PAR1 function, targeting of PAR3 may mitigate the effects of PAR1 in activating endothelial responses such as vascular inflammation.T hrombin generation during thrombosis contributes to the pathophysiology of multiple thrombosis-related diseases such as acute lung injury resulting increased endothelial permeability (1), vascular inflammation (1, 2), and edema formation (for recent reviews, see refs. 3 and 4). These complications are the result of thrombin activation of protease-activated receptor-1 (PAR1) (5). Cleavage of the extracellular N terminus of PAR1 initiates signaling (6) by the concomitant activation of heterotrimeric GTP-binding proteins G␣ q and G␣ 12/13 (7-9). The mechanisms by which PAR1 selectively activates G protein signaling remain unknown. Because the nonsignaling receptor PAR3 is also expressed in endothelial cells (10), we surmised that PAR1 and PAR3 are capable of interacting in such manner as to regulate PAR1 signaling. We found that PAR3 directly dimerizes with PAR1 to induce a specific PAR1/G␣ 13 -binding conformation that favors G␣ 13 activation. We propose a model of PAR1 activation involving the interaction with PAR3, which alters the selectivity of PAR1 for G␣ 13 coupling and promotes endothelial barrier dysfunction. PAR3 functions as an essential allosteric modulator of PAR1 signaling through PAR1/3 dimerization and favors a distinct G␣ 13 -activated downstream pathway. Results PAR3 Knockdown in Endothelial Cells Shifts the Potency of ThrombinActivation of PAR1. Endothelial cells express PAR1, -2, and -3, but not PAR4 (10), and of these only PAR1 and PAR3 are thrombinsensitive (11). To address whether PAR3 alters thrombin signaling in endothelial cells, siRNA knockdown was used to reduce thrombin receptor expression in human pulmonary artery endothelial cells (HPAECs). PAR1 and PAR3 suppression was determined by semiquantitative RT-PCR. siRNA against PAR3 did not affect PAR1 expression, whereas it reduced PAR3 expression 8-fold. Conversely, siRNA against PAR1 only modestly affected PAR3 expression, whereas it suppres...

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Section: Discussionmentioning

confidence: 99%

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

McLaughlin

Patterson

Malik

2007

Proc. Natl. Acad. Sci. U.S.A.

160

164

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“…In some cases the conditioned media were concentrated 20-fold with Nanosep 3K omega spin columns (Pall Life Sciences, Melbourne, Australia) before use. Cells were washed twice with ice-cold PBS, incubated at 4°C for 10 min with lysis buffer, and prepared for electrophoresis as previously described (34). Equal amounts of conditioned medium or cell protein were diluted in a reducing SDS-PAGE sample buffer, heated to 70°C for 10 min, separated on a 4 -12% NuPAGE gel (Invitrogen, Mt Waverley, Australia) and electrotransferred to a polyvinylidene difluoride membrane (Pall Life Sciences).…”

Section: Methodsmentioning

confidence: 99%

“…The method for isolation, culture, and characterization of PTC is described in detail elsewhere (7,18,34). Briefly, the cortical tissue was minced finely, washed several times, and agitated for 20 min at 37°C in Krebs-Henseleit buffer (KHB) containing collagenase type II (1 mg/ml), (Worthington, Freehold, NJ).…”

Section: Methodsmentioning

confidence: 99%

“…Intracellular Ca 2ϩ measurements were performed with the fluorescent ratiometic Ca 2ϩ dye fura-2 (fura-2 AM, Molecular Probes) on cells grown to confluence on black 96-well plates (Perkin Elmer Life and Analytical Sciences, Melbourne, Australia) as previously described (34). An automated injection of agonist was made to give overall concentrations of SLIGKV-NH 2 and VKGILS-NH2 of 50 or 100 M in the corresponding wells.…”

Section: Methodsmentioning

confidence: 99%

“…The fibronectin concentration in the culture supernatant was determined with a sandwich ELISA as previously described (34).…”

Section: Methodsmentioning

confidence: 99%

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Proinflammatory and proliferative responses of human proximal tubule cells to PAR-2 activation

Vesey¹,

Kruger²,

Poronnik³

et al. 2007

American Journal of Physiology-Renal Physiology

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Despite the abundant expression of protease-activated receptor (PAR)-2 in the kidney, its relevance to renal physiology is not well understood. A role for this receptor in inflammation and cell proliferation has recently been suggested in nonrenal tissues. The aims of this study were to demonstrate that human proximal tubule cells (PTC) express functional PAR-2 and to investigate whether its activation can mediate proinflammatory and proliferative responses in these cells. Primary human PTC were cultured under serum-free conditions with or without the PAR-2-activating peptide SLIGKV-NH2 (up to 800 μM), a control peptide, VKGILS-NH2 (200 μM), or trypsin (0.01–100 nM). PAR-2 expression (RT-PCR), intracellular Ca2+ mobilization (fura-2 fluorimetry), DNA synthesis (thymidine incorporation), fibronectin production (ELISA, Western blotting), and monocyte chemotactic protein (MCP)-1 secretion (ELISA) were measured. Trypsinogen expression in kidney and PTC cultures was determined by immunohistochemistry and Western blotting. In the kidney PTC were the predominant cell type expressing PAR-2. SLIGKV-NH2, but not VKGILS-NH2, stimulated a rapid concentration-dependent mobilization of intracellular Ca2+ and ERK1/2 phosphorylation and, by 24 h, increases in DNA synthesis, fibronectin secretion, and MCP-1 secretion. These delayed responses appeared to be independent of ERK1/2. Trypsin produced similar rapid but not delayed responses. Trypsinogen was weakly expressed by PTC in the kidney and in culture. In summary, PTC are the main site of PAR-2 expression in the human kidney. In PTC cultures SLIGKV-NH2 initiates proinflammatory and proliferative responses. Trypsinogen expressed within the kidney has the potential to contribute to PAR-2 activation in certain circumstances.

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Crucial role of the cryptic epitope SLAYGLR within osteopontin in renal crystal formation of mice

Hamamoto

Yasui

Okada

et al. 2011

Journal of Bone and Mineral Research

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Osteopontin plays a crucial role in the formation of renal calcium crystals, which are primarily induced by renal tubular cell injury, especially mitochondrial damage. We have previously shown that the impaired Arg-Gly-Asp (RGD) sequence of osteopontin inhibits renal crystal formation by using OPN-transgenic mice and OPN-knockout (OPN-KO) mice. Here, we investigated the effects of an antimurine osteopontin antibody (35B6-Ab) that specifically reacts with the 162 SLAYGLR 168 sequence, which is exposed by thrombin cleavage and is located adjacent to the RGD sequence, on renal crystal formation. Renal crystals induced by daily administration of glyoxylate over 9 days (from days 1 to 9) in a murine model were sporadically detected in the renal tubular cells at the corticomedullary junction, where thrombin-cleaved osteopontin expression was also coincidentally detected. On days 0, 3, 6, and 9, 35B6-Ab administration inhibited renal crystal formation and induced significant morphological changes in a dose-dependent manner (250, 500, and 1000 mg per mouse). Scanning electron microscopy showed that the crystals in 35B6-Ab-treated mice were aberrantly formed and their density was low; in contrast, the crystals in untreated mice that were not administered 35B6-Ab had a radial pattern of growth (rosette petal-like crystals), and their density was high. Microstructure analysis of renal tubular cells by transmission electron microscopy revealed that untreated mice showed collapsed mitochondria in the flattened cytoplasm of renal tubular cells, unlike the corresponding structures in 35B6-Ab-treated mice, in which renal tubular cell injury was inhibited. In vitro, 35B6-Ab was found to inhibit the attachment of 14 C-labeled crystals to renal tubular culture cells and reduce morphological damage to these cells. We conclude that thrombin-cleaved osteopontin plays an important role in formation of renal calcium crystals and that 35B6-Ab contributes to the remarkable inhibition of early-stage renal crystal formation by preventing renal tubular cell injury and crystal-cell attachment. ß

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Thrombin stimulates proinflammatory and proliferative responses in primary cultures of human proximal tubule cells

Cited by 43 publications

References 67 publications

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

Proinflammatory and proliferative responses of human proximal tubule cells to PAR-2 activation

Crucial role of the cryptic epitope SLAYGLR within osteopontin in renal crystal formation of mice

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