Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in α-actinin-4. This study addresses how FSGS-associated mutant α-actinin-4 may induce GEC injury, focusing on endoplasmic reticulum (ER) stress and metabolism of mutant α-actinin-4 via the ubiquitin-proteasome system. In a model of experimental FSGS induced by expression of an α-actinin-4 K256E transgene in podocytes, we show induction of ER stress, including upregulation of ER chaperones (bip, grp94), phosphorylation of the eukaryotic translation initiation factor-2α subunit, and induction of the proapop totic gene C/EBP homologous protein-10 (CHOP). To address mechanisms of ER stress, we studied signaling in cultured GEC and COS cells expressing α-actinin-4 K256E. Previously, we showed that expression of this α-actinin-4 mutant in GEC increased apoptosis. In the present study, we show that α-actinin-4 K256E upregulates grp94 and CHOP expression in COS cells and significantly exacerbates induction of bip and CHOP in GEC in the presence of tunicamycin. ER stress was associated with aggregation and ubiquitination of α-actinin-4 K256E and impairment of the ubiquitin-proteasome system. In addition, α-actinin-4 K256E exacerbated apoptosis in the context of mild proteasome inhibition. Thus α-actinin-4 K256E triggers several metabolic abnormalities, which may lead to GEC injury and glomerulosclerosis.
- endoplasmic reticulum stress
- transgenic mice
- ubiquitin-proteasome system
the ubiquitin-proteasome system plays an important role in regulating the half-life of short-lived cellular regulatory proteins and in selective degradation of damaged or abnormal proteins. Its function is of major importance to fundamental cellular processes and disease (32). The proteasome may degrade aberrant cytoplasmic or cytoskeletal proteins, as well as misfolded endoplasmic reticulum (ER) proteins, which are retrotranslocated selectively to the cytoplasm. The latter process is known as ER-associated degradation (ERAD) (5, 18). Ubiquitin-proteasome pathway-mediated protein degradation involves tagging of the substrate by covalent attachment of ubiquitin molecules via a three-step reaction and degradation of the tagged protein by the 26S proteasome complex with release of free and reusable ubiquitin. Ubiquitinated proteins are usually degraded efficiently by the proteasome, but sometimes large amounts of misfolded proteins may form aggregates that are covalently linked to each other. Misfolded proteins and/or formation of aggregates may impair the function of the proteasome and may lead to the activation of stress pathways and cytotoxicity (32).
Impairment or “choking” of the ubiquitin-proteasome system may be associated with the exacerbation of ER stress, although the mechanism of this effect is poorly understood (6, 16, 26, 39). Normally, secretory, luminal, and membrane proteins attain their correctly folded conformation in the ER via ER-resident chaperones, and to rescue misfolded proteins, the ER has in place quality control machinery, including the unfolded protein response (UPR) (16, 28, 40) and ERAD. The UPR is a stress response that upregulates the capacity of the ER to process abnormal proteins. Upon accumulation of misfolded proteins in the ER, activating transcription factor-6 moves to the Golgi and is cleaved by proteases. The cleaved cytosolic fragment migrates to the nucleus to activate transcription of ER stress proteins, e.g., the glucose-regulated proteins (grp) grp94 and bip (grp78) and others. In parallel, inositol requiring-1α activates its endoribonuclease activity, cleaving X-box binding protein-1 mRNA and changing the reading frame to yield a potent transcriptional activator. Normally, ER stress proteins serve as protein chaperones for exocytosis from the ER and may complex with defective proteins to target them for degradation. During stress, the induction of ER chaperones may limit accumulation of abnormal proteins. Another aspect of the UPR involves PKR-like ER kinase (PERK), which is activated to phosphorylate the eukaryotic translation initiation factor-2α subunit (eIF2α). This process reduces initiation AUG codon recognition; thus the general rate of translation is reduced (which decreases the protein load on a damaged ER). The UPR may allow cells to recover from stress and may be protective to additional insults, but substantial/prolonged ER stress may lead to apoptosis via induction of specific genes, e.g., C/EBP homologous protein-10 (CHOP; also known as GADD153), and/or activation of caspase-12 or protein kinases (16, 25, 28, 37, 39, 40). The UPR and ERAD are intimately linked, i.e., UPR induction may increase ERAD capacity, and interference with ERAD may lead to UPR induction.
The structure, shape, and biosynthetic capacity of cells is dependent on the actin cytoskeleton. Organization of the actin cytoskeleton is complex. Actin polymerization may be influenced by Rho GTPases and may also be regulated by proteins that bind both actin monomers and phosphatidylinositol 4,5-bisphosphate (e.g., gelsolin, cofilin, α-actinin) (4, 12). Four α-actinin genes encode highly homologous proteins, which form ∼100-kDa head-to-tail homodimers (14). α-Actinins interact with various proteins, e.g., α-actinin-1 may link membrane proteins with the cortical actin cytoskeleton. The best defined function of α-actinin-4 is to cross link/bundle actin filaments, and it may enhance cell motility and elicit tumor-suppressor activity (14).
Glomerular visceral epithelial cells (GEC) or podocytes are intrinsic components of the kidney glomerulus and play a key role in the maintenance of glomerular permselectivity (20, 24, 27, 31). Under normal conditions, GEC are in contact with extracellular matrix and are exposed to trivial concentrations of growth factors, and there appears to be little turnover of GEC. Glomerular permselectivity is dependent on the maintenance of the appropriate structure of GEC and of the filtration slit diaphragms, including nephrin, a key component of the slit diaphragm. Certain forms of glomerulonephritis are associated with GEC injury, which may lead to impaired glomerular function or permselectivity (proteinuria), apoptosis, and glomerulosclerosis. Based on studies in experimental animal models, there is evidence that in focal segmental glomerulosclerosis (FSGS), GEC apoptosis may lead to “podocytopenia” and consequently glomerulosclerosis (17, 21, 35, 36). Alterations in the expression of GEC structural proteins and filtration slit diaphragm components have also been reported (20, 27, 31).
Among the cytoskeletal proteins, α-actinin-4 plays an important role in GEC biology. The human kidney expresses only α-actinin-4 (although mouse GEC also express α-actinin-1) (14, 38). In cultured GEC, α-actinin-4 mediates adhesion and cytoskeletal dynamics (11, 22). Knockout of α-actinin-4 in mouse podocytes in vivo leads to severe glomerular disease, renal failure, and early death (13, 19, 38). Deficiency of α-actinin-4 leading to human disease is not described, but several point mutations (e.g., K255E) or single amino acid deletions in α-actinin-4 are found in heritable forms of human FSGS. Moreover, expression of an α-actinin-4 K256E transgene in mouse GEC in vivo (K256E is the mouse mutant corresponding to human K255E) resulted in development of proteinuric FSGS (23), and homozygous (but not heterozygous) knock-in of the mutant α-actinin-4 allele into α-actinin-4-null mice induced proteinuria (38). These α-actinin-4 mutant proteins show increased binding to actin filaments compared with the wild-type protein (23, 33, 38). Furthermore, by use of biochemical and imaging techniques, it has been demonstrated that an α-actinin-4 mutant form aggregates in cells, that the mutant is unstable and degraded more rapidly, compared with the wild-type protein, and that inhibition of the proteasome attenuates degradation of mutant α-actinin-4 (33, 34, 38). Moreover, expression of the α-actinin-4 K256E mutant in cultured GEC increased apoptosis (3).
The aim of the present study was to further address the signals that regulate GEC injury in vivo and in culture, focusing on α-actinin-4 K256E and ER stress, as well the potential role of the proteasome in the stress response. We show that α-actinin-4 K256E upregulates ER stress in cultured cells and in vivo. ER stress was associated with aggregation and ubiquitination of α-actinin-4 K256E and impairment of the ubiquitin-proteasome system. In addition, α-actinin-4 K256E exacerbated apoptosis.
MATERIALS AND METHODS
Tissue culture reagents and Lipofectamine 2000 were obtained from Invitrogen (Burlington, ON). Pepsin-solubilized bovine dermal collagen I (PureCol) was from Inamed, (Fremont, CA). Rhodamine-conjugated phalloidin, tunicamycin, MG132, and rabbit anti-ubiquitin antiserum were obtained from Sigma-Aldrich Canada (Mississauga, ON). Bisbenzimide H33342 fluorochrome, propidium iodide, and lactacystin were obtained from Calbiochem (La Jolla, CA). Electrophoresis and immunoblotting reagents were from Jackson ImmunoResearch (West Grove, PA), Pall (Pensacola, FL), and GE Healthcare (Baie d'Urfé, Quebec). Rabbit anti-phospho-eIF2α (serine51) was purchased from New England Biolabs (Mississauga, ON). Rabbit anti-bip, rat anti-grp94, and mouse anti-heat shock protein (Hsp) 70 antibodies were from Assay Designs (Ann Arbor, MI). Mouse anti-green fluorescent protein (GFP) and rabbit anti-CHOP antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). pEGFPU was kindly provided by Dr. Ron Kopito (Stanford University, Stanford, CA) (1). Rabbit anti-α-actinin-4 antiserum was produced by immunization with a synthetic peptide corresponding to the first 11 amino acids of the N-terminal region of mouse α-actinin-4, as described (14).
Cell culture and transfection.
Type I collagen gel matrices were prepared as described previously (8). For practical reasons, collagen I was used in all experiments in this study, but in previous studies several key observations were confirmed using collagen IV substrata (2, 8). A rat GEC line (parental GEC) was established from explants of rat glomeruli and has been characterized previously (7, 8). Under standard conditions, parental GEC were cultured on collagen I matrices in K1 medium, which consisted of DMEM/F12 (1:1), containing 5% NuSerum and hormone supplements (8). Production and characterization of GEC that stably express wild-type or mutant GFP-α-actinin-4 were described previously (3). Experiments were done with cells between passages 25 and 45.
COS-1 cells were grown on plastic substratum in DMEM with 10% fetal calf serum (9, 10). COS-1 cells were transiently transfected with plasmid DNAs using the Lipofectamine 2000 reagent, according to the manufacturer's instructions.
Mouse model of FSGS.
A transgenic mouse model of FSGS was developed by expressing murine α-actinin-4 K256E (i.e., a mutation analogous to that affecting a human FSGS family) in a podocyte-specific manner using the murine nephrin promoter. Details of transgene construction and characterization of the animals were published previously (23). Briefly, by 10 wk of age, some of the transgenic mice develop proteinuria and exhibit histological features consistent with FSGS, including segmental sclerosis and tuft adhesion of some glomeruli, tubular dilatation, mesangial matrix expansion, as well as regions of podocyte vacuolization and foot process fusion (23). Control mice consisted of littermates that did not express the nephrin promoter-α-actinin-4 K256E transgene. Mice were studied at 6 mo of age. Glomeruli were isolated by differential sieving (10). The purity of glomerular isolation was ∼65%. The studies were approved by the McGill University Animal Care Committee.
Immunoprecipitation and immunoblotting.
Following cells lysis, samples were adjusted to contain equal amounts of proteins, which were immunoprecipitated with primary antiserum (2). Immune complexes were incubated with agarose-coupled protein A. Complexes were boiled in Laemmli sample buffer and subjected to SDS-PAGE under reducing conditions, as detailed previously (2). For immunoblotting, equal amounts of proteins were loaded into each lane of a gel. Proteins were then electrophoretically transferred onto nitrocellulose paper, blocked with 5% milk, and incubated with a primary antibody, and then with a horseradish peroxidase-conjugated secondary antibody. The blots were developed using the enhanced chemiluminescence (ECL) technique. Quantification of immunoblots was performed by densitometry. Blots were scanned, specific bands of interest were selected, and the density of the bands was measured using National Institutes of Health ImageJ software. Results are expressed in arbitrary units (2, 9, 10). Preliminary studies demonstrated that there was a linear relationship between densitometric measurements and the amounts of protein loaded onto gels.
Measurement of apoptosis and cell number.
Staining with Hoechst H33342 dye was used to quantify apoptosis, as described previously (2). Briefly, adherent cells were stained with H33342 (1 μg/ml) for 10 min at 37°C without fixation. After washing with PBS, cells were stained with propidium iodide (5 μg/ml) to identify necrotic or late apoptotic cells. Cells were examined using a Nikon Diaphot fluorescence microscope, and the number of H33342- and propidium iodide-positive cells was quantified by visual counting. H33342-positive cells (i.e., with condensed and/or fragmented nuclei) without propidium iodide staining were defined as apoptotic. Propidium iodide-positive cells were defined as late apoptotic (2).
Cell number was determined by visual counting (2). Adherent cells were placed into single-cell suspension with trypsin-EDTA. Suspended cells were then counted in a hemacytometer.
Cells adherent to uncoated or collagen-coated glass coverslips were fixed with 3% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100. After washing, cells were incubated with rhodamine-phalloidin (0.043 μg/ml) for 20 min to visualize F-actin. Coverslips were mounted onto glass slides and were examined using both rhodamine and fluorescein filters in a Nikon Diaphot fluorescence microscope. Slides were photographed using a Nikon Coolpix 995 digital camera locked at a constant exposure (3). Quantification of fluorescence intensity and merging of fluorescence images were carried out with Adobe Photoshop software.
Data are presented as means ± SE. The t-statistic was used to determine significant differences between two groups. One-way ANOVA was used to determine significant differences among groups. Where significant differences were found, individual comparisons were made between groups using the t-statistic and adjusting the critical value according to the Bonferroni method.
ER stress is evident in experimental glomerular injury.
Mice expressing an α-actinin-4 K256E transgene in GEC develop proteinuria and FSGS (23). To determine whether GEC injury in vivo is associated with activation of stress pathways, we examined induction of the UPR in this experimental model. Expression of the ER chaperones, bip and grp94, was increased 1.7- to 2.2-fold in glomeruli isolated from transgenic mice compared with control (Fig. 1). Another aspect of the UPR involves phosphorylation of eIF2α on serine51. This process reduces initiation AUG codon recognition, thereby blunting the general rate of translation (which aims at decreasing the protein load on a damaged ER). eIF2α phosphorylation was increased modestly (1.2-fold), but significantly in glomeruli isolated from transgenic mice, compared with control (Fig. 1).
Although phosphorylation of eIF2α is associated with a global decrease in translation, certain mRNAs may be translated preferentially after eIF2α is phosphorylated. Among these is activating transcription factor-4, which induces expression of several genes, including CHOP, a gene closely associated with apoptosis and/or growth arrest (37, 40). Compared with controls, expression of CHOP was enhanced 2.5-fold in transgenic mice (Fig. 1). We also examined expression of the cytosolic stress protein Hsp70. In contrast to the pattern of ER stress induction, expression of Hsp70 was not increased in transgenic mouse glomeruli (Fig. 1). All of the transgenic mice showed moderate to severe albuminuria (data not shown). In summary, in this mouse model of FSGS, ER chaperone expression, eIF2α phosphorylation, and induction of CHOP were increased.
α-Actinin-4 K256E expression facilitates induction of ER stress.
To address the mechanisms of ER stress induction in GEC injury in FSGS, we pursued further studies in cell culture. Abnormal polypeptides are generally detrimental to cellular function, and consequently, stress pathways may be activated. The first set of studies was designed to establish whether the FSGS-associated GFP-α-actinin-4 K256E mutant can influence ER stress. To induce a high level of α-actinin-4 protein expression, we transiently transfected COS cells with GFP-tagged mutant and wild-type α-actinin-4 proteins (Fig. 2, A and B). It should be noted that there was no significant proteolytic degradation of GFP-α-actinin-4 (Fig. 2A). By immunoblotting, expression of GFP-α-actinin-4 wild-type was consistently greater than K256E, and expression of the latter was slightly below endogenous α-actinin-4 (Fig. 2B). This result was confirmed by quantifying the fluorescence intensity of expressed GFP-α-actinin-4 proteins (Fig. 2C). Experiments were carried out 48 h after transient transfection, at which time protein expression reached a maximum. After 48 h, expression of the ER chaperone grp94 increased 2.6-fold, while CHOP increased 1.7-fold in COS cells transfected with GFP-α-actinin-4 K256E compared with wild-type (Fig. 3, A and B), in keeping with ER stress induction by GFP-α-actinin-4 K256E. The levels of grp94 and CHOP in the COS cells transfected with GFP-α-actinin-4 wild-type were comparable to levels in untransfected COS cells (results not shown).
Previously, we produced subclones of cultured GEC that stably express the α-actinin-4 K256E mutant, and we have shown that stable expression of this mutant in GEC increased apoptosis and caspase-3 activity compared with GEC stably expressing GFP-α-actinin-4 wild-type or parental GEC (3). In the next series of experiments, we examined whether expression of α-actinin-4 K256E in GEC was associated with the induction of ER stress. We employed GEC stably transfected with either GFP-α-actinin-4 K256E mutant or wild-type proteins, as well as parental cells for comparison. In keeping with earlier results (3), and by analogy to the transient transfection model in COS cells, there was an approximately threefold higher level of expression of the α-actinin-4 wild-type protein compared with the mutant (Fig. 3C). In resting GEC, expression of the mutant or wild-type α-actinin-4 did not increase expression of the ER chaperone, bip, or CHOP compared with parental GEC (Fig. 3, D–F). Tunicamycin, a drug that blocks N-linked glycosylation and causes an accumulation of misfolded proteins in the ER, is an inducer of ER stress (40). After incubation of GEC with tunicamycin (24 h), the GEC stably transfected with GFP-α-actinin-4 mutant showed increases in bip and CHOP (8- and 27-fold, respectively) that significantly exceeded the increases seen in cells expressing the wild-type protein, as well as the increases in parental GEC (5- and 9- to 12-fold, respectively; Fig. 3, D–F). The effect of GFP-α-actinin-4 K256E on the induction of ER stress by tunicamycin was particularly robust, given that expression of the mutant was substantially lower than the wild-type protein. Thus, while expression of α-actinin-4 K256E in GEC did not independently induce ER stress, the mutant allowed or exacerbated responsiveness to the induction of ER stress by tunicamycin. Tunicamycin did not induce ER stress after only 6 h of incubation.
α-Actinin-4 K256E forms microaggregates.
The above experiments demonstrate that high-level expression of α-actinin-4 K256E (i.e., in COS cells) induced ER stress, while a lower level of expression (in GEC) enhanced susceptibility of cells to the induction of ER stress. To determine how the α-actinin-4 K256E mutant could lead to induction of ER stress, we first focused on the localization of α-actinin-4. GEC expressing GFP-α-actinin-4 wild-type or mutant proteins were stained with rhodamine-phalloidin (to visualize F-actin) and were examined by fluorescence microscopy. In keeping with earlier results (3), F-actin showed a cortical distribution, together with the presence of some stress fibers (Fig. 4). Both the wild-type and mutant GFP-α-actinin-4 showed cortical as well as cytoplasmic localization, and the wild-type protein was also present in the nuclei of some cells (Fig. 4). In contrast, GFP-α-actinin-4 K256E was not found in the nucleus, but in some cells the mutant protein appeared to aggregate (Fig. 4). This result supports the view that GFP-α-actinin-4 K256E is an abnormal protein that may be misfolded, tending to form microaggregates.
α-Actinin-4 K256E induces impairment of the ubiquitin-proteasome pathway.
We hypothesized that α-actinin-4 K256E or its aggregates may be degraded via the ubiquitin-proteasome pathway, and as a result, the pathway may become overwhelmed and functionally impaired. Such choking of the ubiquitin-proteasome system is reported to increase ER stress (16, 39). To verify that this mechanism could operate in GEC, we initially demonstrated that incubation of GEC with two distinct proteasome inhibitors, MG132 and lactacystin, can indeed lead to upregulation of bip and CHOP expression (Fig. 5). Increases in bip expression ranged from 1.2- to 2.8-fold; CHOP increases were 4- to 18-fold. While these increases were not as great as those induced by tunicamycin, these results confirm that proteasome inhibition can enhance ER stress in GEC.
Next, we studied whether degradation of GFP-α-actinin-4 K256E affects the function of the ubiquitin-proteasome pathway. Since these experiments require relatively high levels of α-actinin-4 expression, the studies were carried out using transient transfection in COS cells. The function of the ubiquitin-proteasome system was monitored by a reporter consisting of a short degron, CL1, fused to the C-terminus of GFP (GFPU). GFPU is rapidly degraded when ubiquitin-proteasome function is normal, whereas impairment of the ubiquitin-proteasome system, e.g., via enhanced flux of a mutant/aggregated protein, will reduce degradation of GFPU, resulting in an increased level of GFPU expression (1). COS cells were transiently cotransfected with GFPU (or GFP for comparison), plus K256E mutant or wild-type forms of GFP-α-actinin-4. In the cells transfected with wild-type GFP-α-actinin-4, expression of GFPU was 42% lower than GFP (at 48 h), since GFPU is degraded by the proteasome, but GFP is stable (Fig. 6, A and B). Compared with the wild-type, GFP-α-actinin-4 K256E did not affect GFP expression, but the mutant enhanced GFPU expression (GFPU was only 17% lower than GFP), indicating that degradation of GFPU by the ubiquitin-proteasome system was impaired (Fig. 6, A and B). In COS cells transfected only with GFP or GFPU (i.e., without GFP-α-actinin-4), the expression of GFP and GFPU was similar to the cells cotransfected with GFP-α-actinin-4 wild-type (results not shown). Finally, incubation of COS cells with MG132 increased GFPU expression compared with untreated cells, confirming that GFPU is degraded by the proteasome (Fig. 6C).
To verify that α-actinin-4 K256E undergoes ubiquitination, COS cells were transiently transfected with GFP-α-actinin-4 wild-type or mutant, and cells were incubated in the presence of MG132. Probing of the immunoprecipitated GFP-α-actinin-4 with an antibody to ubiquitin showed that the mutant α-actinin-4 underwent polyubiquitination, but the wild-type did not (Fig. 7A). Despite using a cell culture system that allows for relatively high protein expression, the ubiquitination detectable by immunoblotting was weak. Nevertheless, the result was reproducible in three experiments. Polyubiquitinated forms of mutant α-actinin-4 were not, however, detectable with anti-GFP antibody (Fig. 7A).
The above experiments were carried out in COS cells to achieve high levels of α-actinin-4 expression. To confirm that similar pathways were active in GEC stably transfected with GFP-α-actinin-4 K256E, we compared expression of the mutant GFP-α-actinin-4 in untreated GEC with cells treated with MG132. Incubation of GEC with MG132 resulted in a 2- to 3-fold increase in GFP-α-actinin-4 K256E (Fig. 7B), implying that the mutant protein undergoes ubiquitination and degradation by the proteasome.
α-Actinin-4 exacerbates the effect of mild proteasome inhibition on apoptosis/cell survival.
Previously, we demonstrated that stable expression of the GFP-α-actinin-4 K256E mutant in GEC reduced cell number and increased apoptosis and caspase-3 activity after 72 h of culture compared with GEC stably expressing GFP-α-actinin-4 wild type or parental GEC (3). In the following experiments, COS cells were transiently transfected with the α-actinin-4 K256E mutant or wild-type, or a control plasmid, and were treated with a low concentration of MG132 for 48 h. Compared with untreated cells, MG132 increased the amount of apoptosis; however, apoptosis was enhanced by overexpression of wild-type GFP-α-actinin-4 and was further exacerbated by expression of the mutant (Fig. 8A). At the 48-h time point, both wild-type and K256E GFP-α-actinin-4 had modest independent effects on apoptosis, although the changes were not statistically significant. Nevertheless, in the context of mild proteasome inhibition, expression of α-actinin-4 proteins exacerbates apoptosis markedly. By analogy, treatment of COS cells with MG132 reduced cell number; however, the reduction in cell number was exacerbated markedly by expression of GFP-α-actinin-4 K256E (Fig. 8B).
In recent years, evidence has accumulated to support the view that the early pathogenesis of FSGS is associated with GEC injury, which may then lead to apoptosis (20, 24, 27, 31, 36). Acquired GEC injury may be triggered by immunological factors (e.g., T cell factors affecting permeability), oxidants, human immunodeficiency virus, toxins, detachment of cells from extracellular matrix, and other substances. In addition, FSGS may be associated with heritable mutations in several distinct proteins that play key roles in maintaining GEC ultrastructure, e.g., α-actinin-4. In the present study, we investigated how expression of a mutated cytoskeletal protein may lead to GEC injury in experimental FSGS. In keeping with GEC injury, α-actinin-4 K256E transgenic mice showed albuminuria. Expression of bip, grp94, and CHOP, as well as eIF2α phosphorylation were increased significantly in glomeruli from mice with FSGS, compared with nontransgenic littermate controls (Fig. 1). Bip and grp94 are chaperones involved in normal posttranslational processing of proteins in the ER. Under conditions of stress, where the amount of misfolded protein in the ER increases, induction of the UPR, including the increased production of bip and grp94, may enhance the protein folding capacity. In parallel, phosphorylation of eIF2α by PERK reduces initiation AUG codon recognition, thereby reducing the general rate of translation, which aims at decreasing the protein load on a damaged ER. Prolonged ER stress, however, leads to induction of proapoptotic genes, including CHOP (16, 28, 40). Why some types of ER stress are associated with cytoprotection, whereas others lead to apoptosis is not well established. A recent study showed that after stimulation of cells with low doses of compounds that induce ER stress, mRNAs for bip and CHOP were induced, but while the bip mRNA was stable and persisted, the CHOP mRNA was unstable and decayed (29). In this context, the UPR was cytoprotective. After stimulation with the same compounds at high doses, CHOP mRNA became more stable, and consequently, the UPR may switch to a proapoptotic outcome. Based on our results, it would be reasonable to propose that in the α-actinin-4 K256E model of FSGS, there is pronounced ER stress, which may be contributing, at least in part, to GEC apoptosis. Other forms of glomerulopathies associated with GEC injury have also shown an upregulation of the UPR, including the passive Heymann model of membranous nephropathy in the rat (9, 10). Expression of ER chaperones in GEC was also increased in a rat glomerulopathy induced by excessive protein accumulation due to expression of a megsin transgene (15). However, the mechanisms and consequences of ER stress induction in these two glomerulopathies appear to be distinct from the FSGS model in this study.
To better understand the mechanisms of ER stress induction associated with α-actinin-4 K256E expression, we examined biochemical alterations induced by α-actinin-4 K256E in cultured cells. Previously, we have shown that stable expression of the α-actinin-4 K256E mutant protein in GEC reduced cell number and increased apoptosis and caspase-3 activity, compared with GEC stably overexpressing α-actinin-4 wild type, or parental GEC (2). Interestingly, by fluorescence microscopy, the GFP-α-actinin-4 mutant appeared to form microaggregates in GEC (Fig. 4), suggesting that this abnormal protein may be misfolded, leading to aggregation. This result is in keeping with earlier studies, which showed an analogous result in mouse GEC, and which employed fluorescence microscopy and/or density-gradient centrifugation to show that α-actinin-4 mutants can form high molecular mass aggregates (33, 34, 38). Also, the α-actinin-4 mutant was degraded more rapidly in cells, compared with α-actinin-4 wild-type. After transfection in GEC or COS cells, expression of α-actinin-4 K256E was consistently lower than the wild-type protein, suggesting that there may have been ongoing degradation of the mutant protein (Figs. 2 and 3). Based on these observations, it is reasonable to propose that α-actinin-4 K256E, or aggregates of this protein may induce a stress response, and this response may be due to ubiquitination and degradation via the ubiquitin-proteasome pathway, such that the pathway becomes overwhelmed and functionally impaired (32). Ubiquitin conjugation or cleavage by caspases may also reduce the protein's solubility, thus favoring formation of aggregates. Transfection of COS cells with α-actinin-4 actually resulted in ubiquitination of the K256E but not wild-type protein (Fig. 7). Furthermore, using GFPU as a reporter of proteasome function (1), we confirmed that expression of α-actinin-4 K256E can impair the ubiquitin-proteasome system (Fig. 6). For technical reasons, these experiments required relatively high levels of α-actinin-4 expression, and were, therefore, carried out using transient transfection in COS cells. However, as the experiments focus primarily on biochemical aspects of a mutated protein, it is probable that the same mutant protein would be similarly ubiquitinated and processed by the proteasome in other cell types. Indeed, inhibition of the proteasome increased the level of α-actinin-4 K256E expression in GEC (Fig. 7) (38), thereby indicating that it is likely subject to proteasomal degradation.
α-Actinin-4 is a cytosolic/cytoskeletal protein that would not normally enter the ER to undergo posttranslational modification, but, nonetheless, levels of grp94 and CHOP were greater in COS cells transiently transfected with the α-actinin-4 mutant compared with wild-type (Fig. 3), indicating that high-level expression of α-actinin-4 K256E can induce ER stress. Furthermore, after stable expression of α-actinin-4 K256E in GEC, these cells showed enhanced susceptibility to the induction of ER stress by tunicamycin (Fig. 3). Thus tunicamycin had a modest and insignificant effect on the induction of CHOP in GEC expressing wild-type α-actinin-4 and in parental GEC but induced CHOP expression substantially in the presence of α-actinin-4 K256E. These results indicate that while stable expression of α-actinin-4 K256E in GEC did not induce the UPR directly, the mutant most likely affected the integrity of the ER adversely, which may render these cells more susceptible to additional stress and induction of proapoptotic genes. Both the present (Fig. 5) and earlier studies have shown that exacerbation of ER stress can be associated with impairment of the ubiquitin-proteasome system (6, 16, 26, 39). The mechanism is poorly understood (39), and it may involve proteasome subunits that are associated with the ER, or interference with ERAD. Moreover, in COS cells, expression of α-actinin-4 K256E exacerbated apoptosis in the context of mild proteasome inhibition (Fig. 8), further supporting the view that apoptosis associated with mutant α-actinin-4 may involve induction of ER stress via proteasome inhibition.
The pathogenesis of FSGS due to α-actinin-4 K256E may resemble processes in certain age-related or neurodegenerative diseases involving proteins with polyglutamine repeats, such as Parkinson's, Huntington's, and Alzheimer's diseases (26, 30, 32, 38, 39). However, the mechanistic relationships between abnormal proteins and cell injury require further study. As stated above, misfolded proteins induce dysfunction of the ubiquitin-proteasome system, i.e., the misfolded proteins “choke” or “gum up” the proteasome, and this process may increase stress in cellular compartments, including the ER. Furthermore, ubiquitination regulates many essential cellular processes, including normal protein degradation, cell cycle, transcription, DNA repair, and protein trafficking; consequently, disrupted ubiquitin signaling may have broad adverse consequences for cell function. Finally, misfolded proteins that aggregate could sequester other important cellular proteins into the aggregates, thereby impairing the ability of these proteins to mediate key cellular processes. By analogy to experimental neurodegenerative disease models, while our results in cell culture are intriguing, one must be cautious in extrapolating the cell culture events, which are based on overexpression and a relatively brief experimental time frame, to a disease process that in humans takes many years to become established.
One also needs to consider the possibility that the pathological effect of α-actinin-4 K256E may be due, at least in part, to cytoskeletal disruption (34). Normally, α-actinin-4 may be required for integrin-dependent adhesion of GEC (11), and GEC that express α-actinin-4 K256E show defective spreading and motility (22). Further studies, including in vivo models, will be required to ascertain the functional importance of the proteasome and ER stress-associated pathways in the pathogenesis of GEC injury and FSGS.
This work was supported by Research Grants from the Canadian Institutes of Health Research (MOP-84213, MOP-53264, MOP-53335) and the Kidney Foundation of Canada. A. V. Cybulsky was supported by a Scholarship from the Fonds de la Recherche en Santé du Québec and the Catherine McLaughlin Hakim Chair. T. Takano holds a Scholarship from the Fonds de la Recherche en Santé du Québec.
We thank Dr. Simon Wing (McGill University) for helpful discussions.
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