Figure 1.
Schematic of the signal transduction involving MK2, MK3, and MK5.
Activation of the p38 MAPK by various stress stimuli or growth factors results in activation of MK2 and MK3. The signal transduction by these protein kinases towards the major substrates tristetraprolin (TTP) and HSPB1 is typically additive, although in most cells MK2 is the prevailing signal transducer, with little contribution of MK3. MK5 is activated by PKA, probably independently of p38 MAPK. The role of putative MK5 becomes obvious in the absence of MK2 and MK3, as shown in the present study. MK2, MK3, and MK5 phosphorylate the same two sites in mouse HSPB1 (Ser15, Ser86). In addition to MK2 and MK3, MNK1 and MSK1/2 are further MKs that are downstream of p38 MAPK. In macrophages, MK2 and MK3 were found to control expression of the immune response mediators IFNβ, IL-10, and NFκB through regulation of the activity of IRF3 and IκBβ. In these cells, MK2 was demonstrated to prevent MK3 from exerting negative regulatory effects on IRF3- and NFκB-dependent signaling. Dashed arrows indicate indirect signal transduction, while open arrows indicate complex effects on biological responses.
Figure 2.
Survival rate of mice with disrupted MK2 and/or MK3 genes following injection with AMC serum.
13 mice from each group were given 100 µl of either control serum or AMC serum by i.v. injections for 4 consecutive days (days 1–4). At day 8, one or two mice from each group with pronounced disease symptoms were sacrificed for tissue harvesting (open arrow). These mice were included in the mortality count. At day 16, one or two mice from each group with no obvious disease symptoms were also sacrificed (closed arrow). These mice were excluded from the mortality count. *Log rank test.
Figure 3.
Effect of MK2 and MK3 genotypes on proteinuria in response to the AMC serum.
(A) Scatter plots show the urinary protein/creatinine ratios from samples collected from all mice at day 0 prior to AMC serum injection and at days 4, 8, and 12 following AMC serum injection. Horizontal bars indicate the means and error bars represent S.D. Asterisks indicate significant (P<0.05) differences between means, as compared to the wild-type group at the same day. At days 8 and 12, all means were significantly greater than baseline proteinuria values at day 0 of the same genotypic group, with exception of the MK2/MK3 double knock-out (MK2−/−MK3−/−) mice at day 8. Given the high degree of variability within each experimental group, potential differences in mean proteinuria values among all other groups failed to reach statistical significance. Urine samples of mice selected for electrophoretic protein analysis as shown in (B) are labeled by numbers in the panels of days 4, 8, and 12. Note that in some instances the amount of collected urine was not sufficient for protein determination (i.e. the number of dots is less than the number of surviving mice as shown in Figure 2). (B) Urinary serum albumin excretion of selected mice (numbered 1 - 20) from the different MK2 and MK3 genotypes as visualized on Coomassie-stained SDS gels. Some of the selected mice survived throughout the entire experiment, while others died after day 4. The mouse numbers correspond to the numbered proteinuria values as indicated in (A). Consistent with the protein/creatinine ratios shown in (A), at day 0 mice of all genotypes had negligible albuminuria. Following AMC serum treatment, massive albuminuria was detected in most of the mice, with some variation in its extent and onset.
Figure 4.
Cortical morphological lesions from wild-type and MK2/MK3 knock-out mice in response to the AMC serum.
Kidney sections from untreated and AMC serum-treated wild-type mice (MK2+/+MK3+/+), MK2/MK3 double knock-out mice (MK2−/−MK3−/−), and MK3 knock-out mice (MK2+/+MK3−/−) on day 16 are shown. (A) Periodic acid-Schiff stain of the renal parenchyma of wild-type (panels a, c) and MK2/MK3 double knock-out mice (panels b, d), left untreated (panels a, b) or treated with the AMC serum (panels c, d). Normal morphology of the renal parenchyma was noted in untreated mice of both genotypes. Renal injury in response to the AMC serum included dilation of renal tubules and the presence of hyaline casts. Arrows designate glomeruli. Scale bar: 100 µM. (B) Silver stain (panels a–c, e) and trichrome stain (panels d, f) of glomeruli from untreated wild-type (panel a) and MK2/MK3 double knock-out mice (panel b), and from AMC serum-treated MK2/MK3 double knock-out (panels c–e) and MK3 knock-out mice (panel f). A preserved glomerular morphology was noted in untreated wild-type and MK2/MK3 double knock-out mice. Glomerular injury in response to the AMC serum included a thickening of the capillary walls due to duplication of basement membranes (tram-tracking) with associated mesangial interposition and narrowing of the capillary lumina (panel c, arrows), small fuchsinophilic subendothelial and mesangial deposits (panel d, arrows), and necrotizing lesions with associated crescent formation (panel e). These lesions were noted in mice of all genotypes. In addition, large, wire-loop type subendothelial deposits were found in the MK3 knock-out mice (panel f, arrows). Scale bar: 50 µM.
Figure 5.
p38 MAPK→MK2/MK3→HSPB1 signaling and stress response in MK2/MK3 knock-out mice following AMC serum treatment.
Extracts of renal cortices were processed for IEF-PAGE (A, panel a) or SDS-PAGE (A, panels b, c; B, C, D) from untreated mice (day 0; baseline control) and AMC serum-treated mice (day 8 of treatment). (A) Phosphorylation, baseline expression and induction of HSPB1. Panel a shows the distribution of the various HSPB1 isoforms (0p, unphosphorylated; 1p, singly phosphorylated; 2p, doubly phosphorylated) within each sample. Sample loading aimed to obtain comparable overall signals, in spite of considerable differences in the total HSPB1 content among the samples (cf. panel c). Panel b shows the amounts of Ser86-phosphorylated HSPB1 (p-Ser86). Equal amounts of total protein (15 µg) were loaded onto each lane. Panel c shows baseline expression and induction of HSPB1 in response to the AMC serum. (B) Baseline expression and response to the AMC serum of the heat shock proteins, HSPB6, HSPB8, and HSP70 (panels a–c, respectively). (C) Expression or phosphorylation of indicators of the unfolded protein response, CHOP (panel a), GRP78 (panel b), and eIF2α (panels c, d), before and after AMC serum treatment. Panels c and d show phosphorylated (p-eIF2α) and total eIF2α, respectively. (D) Expression of β-actin served as a loading control. Overall, this figure demonstrates partial involvement of MK2 and MK3 in baseline expression and/or phosphorylation of a number of sHSPs and indicators of the unfolded protein response, as well as in their pathophysiological response following AMC serum treatment.
Figure 6.
Distribution of HSPB1 in renal cortices in response to the AMC serum.
Paraffin-embedded renal cortices of untreated and AMC serum-treated wild-type and MK2/MK3 double knock-out mice (day 16 following AMC serum treatment) were sectioned and processed for immunofluorescence microscopy. Total HSPB1 was visualized using an anti-HSPB1 antibody. In untreated control mice of either genotype, labeling of the glomeruli (including Bowman's space) was moderately elevated as compared to the surrounding tubules (upper row, left panels) or to the more distant tubules (upper row, right panels). AMC serum treatment caused a strong increase in HSPB1 labeling in the tubules, both adjacent to the glomeruli (lower row, left panels) and more distant from the glomeruli (lower row, right panels), thus indicating a stress response in the tubular compartment.
Figure 7.
Expression and phosphorylation of various MKs in renal cortices in response to the AMC serum.
Extracts of renal cortices were processed for SDS-PAGE from untreated mice (day 0; baseline control) and AMC serum-treated mice (day 8 of treatment). Expression and phosphorylation (activation) of MK5 (A), MNK1 (B), and MSK1 (C) before and after AMC serum treatment are shown. The dot in (B) marks the correct MNK1 band (upper band; ∼48 kDa), whereas the lower band (∼44 kDa) probably results from an unspecific cross-reaction of the antibody. (D) Expression of GAPDH served as a loading control.
Table 1.
Summary of the observed baseline regulation in renal cortices with various MK2 and MK3 genotypes.
Table 2.
Summary of the observed regulation in response to the AMC serum in renal cortices with various MK2 and MK3 genotypes.