Fluid shear stress induces renal epithelial gene expression through polycystin-2-dependent trafficking of extracellular regulated kinase

Abstract

Background: The cilium and cilial proteins have emerged as principal mechanosensors of renal epithelial cells responsible for translating mechanical forces into intracellular signals. Polycystin-2 (PC-2), a cilial protein, regulates flow/shear-induced changes in intracellular Ca(2+) ([Ca(2+)](i)) and recently has been implicated in the regulation of mitogen-activated protein (MAP) kinases. We hypothesize that fluid shear stress (FSS) activates PC-2 which regulates MAP kinase and, in turn, induces MAP kinase-dependent gene expression, specifically, monocyte chemoattractant protein-1 (MCP-1).

Methods: To test this, PC-2 expression was constitutively reduced in a murine inner medullary collecting duct (IMCD3) cell line, and the expression of FSS-induced MCP-1 expression and MAP kinase signaling compared between the parental (PC-2-expressing) and PC-2-deficient IMCD3 cells.

Results: FSS induces MAP kinase signaling and downstream MCP-1 mRNA expression in wild-type IMCD3 cells, while inhibitors of MAP kinase prevented the FSS-induced MCP-1 mRNA response. In contradistinction, FSS did not induce MCP-1 mRNA expression in PC-2-deficient cells, but did increase activation of the upstream MAP kinases. Wild-type cells exposed to FSS augmented the nuclear abundance of activated MAP kinase while PC-2-deficient cells did not.

Conclusions: PC-2 regulates FSS-induced MAP kinase trafficking into the nucleus of CD cells.

Fig. 1

Generation of PC-2-deficient murine IMCD3 cells. a A siRNA oligonucleotide directed against PKD2 (siPKD2) was ligated into a lentivirus vector. A control vector was generated with a siRNA directed against luciferase (siLuc). After transduction and selection, IMCD3 cells containing siPKD2 and siLuc lentivirus vector were isolated. b Western blot of untransduced, VIRHD/P/siLuc transduced, and VIRHD/ P/siPKD2 transduced IMCD3 cells demonstrate that the VIRHD/P/siPKD2 IMCD3 cells express ∼20% PC-2 protein compared to untransduced and control transduced cells.

Fig. 2

MCP-1 mRNA expression in IMCD3 cells exposed to FSS. IMCD3 cells exposed to 0.4 dyn/cm² of shear for 2 h were compared to static controls. FSS-induced MCP-1 (* p < 0.05 compared to static) and did not affect TGF-β₁ and t-PA mRNA expression.

Fig. 3

FSS induces ERK phosphorylation. a Protein lysates of IMCD3 cells exposed to shear or maintained under static conditions were immunoblotted with anti-pERK and anti-total ERK antibodies. Sheared cells expressed abundant pERK at all time points while cells maintained under static conditions did not. b Densitometric ratios of pERK to total ERK were measured in sheared and unsheared IMCD3 cells at 10, 30, 60, and 120 min. Sheared cells express significantly more pERK than static control cells (* p < 0.05 compared to static control).

Fig. 4

FSS induces JNK phosphorylation. a Protein lysates of IMCD3 cells exposed to shear or maintained under static conditions were immunoblotted with anti-pJNK and anti-total JNK antibodies. Sheared cells expressed pJNK at 30, 60, and 120 min while cells maintained under static conditions did not.b Densitometric ratios of pJNK to total JNK were measured in sheared and unsheared IMCD3 cells at 10, 30, 60, and 120 min. Sheared cells express significantly more pJNK than static control cells only at 60 and 120 min (* p < 0.05).

Fig. 5

MAP kinase inhibition reduces FSS-induced MCP-1 expression in wild-type IMCD3 cells. a IMCD3 cells were exposed to 10 μM U0126 under static and shear (0.4 dyn/cm²) conditions, and MCP-1 mRNA was expressed as fold change compared to untreated static control. U0126 significantly reduced MCP-1 mRNA expression in static compared to untreated static cells and abolished the shear-induced increase in MCP-1 (* p < 0.05 compared to untreated static cells). U0126-treated sheared cells expressed greater quantities of MCP-1 mRNA than U0126-treated static controls (^# p < 0.05). b SP600125 (30 μM) treatment of static cells reduced expression of MCP-1 mRNA by 45%, but did not prevent the shear-induced increase in expression of MCP-1 (^# p < 0.05 compared to static control). SP600125 did reduce MCP-1 mRNA expression compared to untreated sheared cells (* p < 0.05), while SP600125-treated cells exposed to shear increased MCP-1 mRNA expression compared to SP600125-treated static cells (⁺ p < 0.05).

Fig. 6

FSS-mediated MCP-1 mRNA expression is abrogated in PC-2-deficient cells. PC-2-deficient IMCD3 cells were exposed to 0.4 dyn/cm² of FSS (n = 6) or maintained under static (n = 6) conditions for 2 h, and MCP-1 mRNA expression measured and compared between each treatment group. As opposed to parental IMCD3 cells, FSS did not induce MCP-1 mRNA in PC-2-deficient cells.

Fig. 7

FSS equally induces ERK (a) and JNK (b) in wild-type and PC-2-deficient cells. Parental and PC-2-deficient IMCD3 cells were exposed to FSS at 0.4 dyn/cm² or maintained under static conditions, and pERK and pJNK expression measured. a IMCD3 (n = 6) and PC-2-deficient (n = 6) cells increased pERK expression upon exposure to FSS. b Similarly, IMCD3 (n = 3) and PC-2-deficient (n = 3) cells also equally increase pJNK expression after exposure to FSS.

Fig. 8

Cilia are expressed in parental and PC-2-deficient IMCD3 cells. Wild-type and PC-2-deficient cells were labeled with a murine monoclonal anti-acetylated-α-tubulin antibody (green) to localize cilia in each cell type. IMCD3(a) and PC-2-deficient (b)cells each expressed a single cilium on the surface of each cell. No gross ciliary aberrations were noted in the PC-2-deficient cells.

Fig. 9

FSS induces nuclear pERK expression in wild-type IMCD3 cells but not in PC-2-deficient cells. Parental and PC-2-deficient IMCD3 cells were exposed to FSS, nuclear (N) and cytoplasmic (C) fractions isolated, and immunoblotted with an anti-phospho-ERK antibody. FSS-induced nuclear expression of pERK in IMCD3 cells (in 3 of 4 experiments), but did increase nuclear pERK in PC-2-deficient cells (in 3 of 3 experiments). GAPDH, a cytoplasmic marker, demonstrates there is no cross-contamination of cytoplasmic fraction into nuclear fraction.