PKC -mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells (original) (raw)
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Journal of Clinical Investigation, 1998
Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [ 125 I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC 50 6.9 nM) and LY294002 (IC 50 6.5 M) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit ( ⌬ p85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or ⌬ p85,
International Journal of Molecular Sciences, 2022
Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1+ endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a p...
American journal of physiology. Renal physiology, 2003
The kidney medulla is physiologically exposed to variations in extracellular osmolality. In response to hypertonic cell shrinkage, cells of the rat kidney medullary thick ascending limb of Henle's loop undergo p38 kinase-dependent regulatory volume increase (RVI). In the present study, we investigated the role of actin cytoskeleton reorganization in this process. Addition of hyperosmotic NaCl or sucrose, which activates MAP kinases and reduces cellular volume, induced a sustained actin polymerization occurring after 10 min and concurrently with RVI. In contrast, hyperosmotic urea, which does not modify MAP kinase activity and cellular volume, did not induce sustained actin polymerization. Fluorescence microscopy revealed that hyperosmotic NaCl and sucrose, but not urea, induced the redistribution of F-actin from a dense cortical ring to a diffuse network of actin bundles. Stabilization of actin filaments by jasplakinolide and inhibition of the generation of new actin filaments b...
Sgk-1 is a Positive Regulator of Constitutive Albumin Uptake in Renal Proximal Tubule Cells
Cellular Physiology and Biochemistry, 2012
Background/Aims: Receptor-mediated endocytosis of albumin by the renal proximal tubule requires a number of proteins including megalin/cubilin, sodium/hydrogen exchanger isoform 3 (NHE3) and ClC-5, as well as the PSD-95/Dlg/Zo-1 (PDZ) scaffold sodium/hydrogen exchanger regulatory factor 2 (NHERF2). Despite members from the AGC kinase family, v-Akt Murine Thymoma Viral Oncogene (Akt or Protein Kinase B) and Serum/Glucocorticoid regulated Kinase 1 (Sgk-1) regulating a number of essential proteins in the albumin handling pathway, their role in uptake is largely unknown. Methods: Opossum kidney (OK) cells were exposed to Texas-Red albumin, in the presence of silencing constructs against Sgk-1, Akt and NHERF2, in addition to the NHE3 inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and NHE3 activator dexamethasone. Target protein was also measured by Western blot analysis in OK cells following exposure to dexamethasone and albumin. Results: Silencing Sgk-1 or overexpression of a dominant negative mutant (DN-Sgk-1) led to a significant reduction of albumin endocytosis compared to control. Conversely, over-expression of wildtype (WT) or constitutively active (CA) Sgk-1 significantly increased uptake. Previous reports have shown Sgk-1 can activate NHE3 through an interaction mediated by NHERF2. We found that silencing both Sgk-1 and NHERF2 demonstrated no additive effect on uptake, suggesting signaling via similar endpoints. Treatment with dexamethasone increased Sgk-1 protein levels and increased
PKCα diffusion and translocation are independent of an intact cytoskeleton
Scientific Reports, 2017
Translocation of cytosolic cPKC to the plasma membrane is a key event in their activation process but its exact nature is still unclear with particular dispute whether sole diffusion or additional active transport along the cell's cytoskeleton contributes to cPKC's dynamics. This was addressed by analyzing the recruitment behavior of PKCα while manipulating the cytoskeleton. Photolytic Ca 2+ uncaging allowed us to quantify the kinetics of PKCα redistribution to the plasma membrane when fused to monomeric, dimeric and tetrameric fluorescence proteins. Results indicated that translocation kinetics were modulated by the state of oligomerization as expected for varying Stokes' radii of the participating proteins. Following depolymerization of the microtubules and the actin filaments we found that Ca 2+ induced membrane accumulation of PKCα was independent of the filamentous state of the cytoskeleton. Fusion of PKCα to the photo-convertible fluorescent protein Dendra2 enabled the investigation of PKCα-cytoskeleton interactions under resting conditions. Redistribution following spatially restricted photoconversion showed that the mobility of the fusion protein was independent of the state of the cytoskeleton. Our data demonstrated that in living cells neither actin filaments nor microtubules contribute to PKCα's cytosolic mobility or Ca 2+-induced translocation to the plasma membrane. Instead translocation is a solely diffusion-driven process.
1999
Song, Jaekyung Cecilia, Bruce J. Hrnjez, Omid C. Farokhzad, and Jeffrey B. Matthews. PKC-e regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS. Am. J. Physiol. 277 (Cell Physiol. 46): C1239–C1249, 1999.—Protein kinase C (PKC) and the actin cytoskeleton are critical effectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the apical or basolateral membrane is poorly defined. In the present study, phorbol 12-myristate 13-acetate (PMA) and other activators of PKC selectively enhanced basolateral but not apical fluidphase endocytosis in human T84 intestinal epithelia. Stimulation of basolateral endocytosis was blocked by the conventional and novel PKC inhibitor Gö-6850, but not the conventional PKC inhibitor Gö-6976, and correlated with translocation of the novel PKC isoform PKC-e. PMA treatment induced remodeling of basolateral F-actin. The actin disassembler cytocha...
BMC Cell Biology, 2009
Background Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled in vitro by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium. Results Exposure of HPAF-II human pancreatic adenocarcinoma cell monolayers to either OI-V or 12-O-tetradecanoylphorbol-13-acetate caused rapid disruption and internalization of AJs and TJs. Activity of classical PKC isoenzymes was responsible for the loss of cell-cell contacts which was accompanied by cell rounding, phosphorylation and relocalization of the F-actin motor nonmuscle myosin (NM) II. The OI-V-induced disruption of AJs and TJs was prevented by either pharmacological inhibition of NM II with blebbistatin or by siRNA-mediated downregulation of NM IIA. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho-associated kinase (ROCK) II, but was insensitive to blockage of MLCK, calmodulin, ERK1/2, caspases and RhoA GTPase. Conclusion Our data suggest that stimulation of PKC disrupts epithelial apical junctions via ROCK-II dependent activation of NM II, which increases contractility of perijunctional actin filaments. This mechanism is likely to be important for cancer cell dissociation and tumor metastasis.
Nephrology Dialysis Transplantation, 2002
Background. Proteinuric renal disease is associated with accumulation of tubulointerstitial matrix proteins. Human proximal tubular cells (PTCs) produce fibronectin in response to serum proteins but not albumin alone. It has been suggested that renal toxicity of filtered albumin depends on its lipid moiety. We therefore investigated the functional consequences of different fatty acids (FAs) carried on human albumin after exposure to human PTCs in culture. Methods. Confluent human PTCs were exposed to recombinant human serum albumin (rHSA) or palmitate (P)-, stearate (S)-, oleate (O)-, and linoleate (L)complexed rHSA. In all experimental conditions, test media contained 1 mguml rHSA alone or carrying 100 mmol FAs. Mitogenic response was assessed by [ 3 H]thymidine incorporation. Cell culture supernatants were assayed for fibronectin. Protein kinase C (PKC) activity was assessed in cell lysates. Results. Apical exposure to rHSA alone or the O-rHSA complex stimulated a significant increase in [ 3 H]thymidine incorporation, whereas the L-rHSA complex was markedly inhibitory to human PTC growth. The L-rHSA complex was associated with severe cytotoxicity as assessed by lactate dehydrogenase release. Among all conditions, O-rHSA was the only test media that significantly increased fibronectin levels over control conditions (150.1"10.6% over control, P-0.05, ns3). Pre-treatment of PTCs with PKC inhibitors before O-rHSA exposure resulted in a dose-dependent decrease in fibronectin secretion. O-rHSA activated PKC significantly compared with controls.
AJP: Cell Physiology, 2005
We investigated the involvement of PKC-ε in apical actin remodeling in carbachol-stimulated exocytosis in reconstituted rabbit lacrimal acinar cells. Lacrimal acinar PKC-ε cosedimented with actin filaments in an actin filament binding assay. Stimulation of acini with carbachol (100 μM, 2–15 min) significantly ( P ≤ 0.05) increased PKC-ε recovery with actin filaments in two distinct biochemical assays, and confocal fluorescence microscopy showed a significant increase in PKC-ε association with apical actin in stimulated acini as evidenced by quantitative colocalization analysis. Overexpression of dominant-negative (DN) PKC-ε in lacrimal acini with replication-defective adenovirus (Ad) resulted in profound alterations in apical and basolateral actin filaments while significantly inhibiting carbachol-stimulated secretion of bulk protein and β-hexosaminidase. The chemical inhibitor GF-109203X (10 μM, 3 h), which inhibits PKC-α, -β, -δ, and -ε, also elicited more potent inhibition of car...
American Journal of Physiology-Cell Physiology, 1999
Protein kinase C (PKC) and the actin cytoskeleton are critical effectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the apical or basolateral membrane is poorly defined. In the present study, phorbol 12-myristate 13-acetate (PMA) and other activators of PKC selectively enhanced basolateral but not apical fluid-phase endocytosis in human T84 intestinal epithelia. Stimulation of basolateral endocytosis was blocked by the conventional and novel PKC inhibitor Gö-6850, but not the conventional PKC inhibitor Gö-6976, and correlated with translocation of the novel PKC isoform PKC-ε. PMA treatment induced remodeling of basolateral F-actin. The actin disassembler cytochalasin D stimulated basolateral endocytosis and enhanced stimulation of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin and jasplakinolide. PMA induced membrane-to-cytosol redistribution of ...