Cytoskeletal keratin glycosylation protects epithelial tissue from injury

Abstract

Keratins 8 and 18 (K8 and K18) are heteropolymeric intermediate filament phosphoglycoproteins of simple-type epithelia. Mutations in K8 and K18 predispose the affected individual to liver disease as they protect hepatocytes from apoptosis. K18 undergoes dynamic O-linked N-acetylglucosamine glycosylation at Ser 30, 31 and 49. We investigated the function of K18 glycosylation by generating mice that overexpress human K18 S30/31/49A substitution mutants that cannot be glycosylated (K18-Gly(-)), and compared the susceptibility of these mice to injury with wild-type and other keratin-mutant mice. K18-Gly(-) mice are more susceptible to liver and pancreatic injury and apoptosis induced by streptozotocin or to liver injury by combined N-acetyl-D-glucosaminidase inhibition and Fas administration. The enhanced apoptosis in the livers of mice that express K18-Gly(-) involves the inactivation of Akt1 and protein kinase Ctheta as a result of their site-specific hypophosphorylation. Akt1 binds to K8, which probably contributes to the reciprocal hyperglycosylation and hypophosphorylation of Akt1 that occurs on K18 hypoglycosylation, and leads to decreased Akt1 kinase activity. Therefore, K18 glycosylation provides a unique protective role in epithelial injury by promoting the phosphorylation and activation of cell-survival kinases.

Figure 1. K18 Gly⁻ predisposes transgenic mice to STZ-induced injury

a, Posttranslational modifications of K18. All IFs consist of three structural motifs (not drawn to scale): a central relatively conserved α-helical “rod” domain that is flanked non-conserved “head” and “tail” domains. Three glyco-serines (gS30/31/49) and two phospho-serines (pS34/53) are located in the head domain, while two caspase digestion sites (D238/397) are located in the rod and tail domains. b, Livers were obtained from non-transgenic (N, lane 1), or transgenic mice that overexpress hK18 WT (lanes 2,3) or Gly⁻ (Ser30/31/49→Ala) (lanes 4,5). Each lane represents separate livers that were used to generate a highly enriched keratin fraction by high salt extraction (HSE) or that were solubilized in SDS-containing sample buffer to prepare total lysates. The samples were separated by SDS-PAGE then stained with Coomassie blue, or were blotted with antibodies specific to hK18 (DC10), mouse/human (m/h) K18 (EndoB), mK8 (Troma-I), or nonphospho-K18 S33 (antibody 4668) that recognizes the hK18 WT sequence ²⁶RPVSSAASVYAGA, but not the K18 Gly⁻ because of the underlined mutant serines. c, In vitro galactosylation of K8/K18. K8/K18 immuno-precipitates from livers similar to those used in panel (b) were obtained by using the human K18-specific antibody L2A1 and then subjected to in vitro galactosylation using UDP-[4,5-³H]-galactose and galactosyltransferase to radiolabel K8/K18 Ser/Thr-linked O-GlcNAcs. Note that mutation of the three K18 S30/31/49 glycosylation sites abolished K18 glycosylation in the Gly⁻ livers. d, In vitro [³H]-galactosylation of detergent-free cytosolic extracts isolated from transgenic K18-WT and K18-Gly⁻ livers 2 days after STZ administration. Equal amounts of protein were separated by SDS-PAGE followed by Coomassie staining or fluorography to assess the extent of protein O-GlcNAc modification. e, f, The indicated mouse genotypes [total number of mice/genotype are shown in panel (f); all in an FVB background] were injected with STZ then monitored daily for survival. Most deaths occurred within 10 days and no deaths were observed beyond 24 days. Mortality was significantly increased in the Gly⁻ mice as compared with other mice strains (~70% Gly⁻ versus ~20–40% in the other listed control strains, p=0.0002).

Figure 2. Comparison of the STZ-induced tissue injury in K18 WT versus K18 Gly⁻mice

a, b, Gross appearance (a) and histopathology (b) of mouse organs were compared before and 2 days after STZ injection. E, edema; H, hemorrhage; I, islet; K, kidney; L, liver; P, pancreas; S, spleen. Bars = 200 μm (panel b). See supplemental Fig S3 for higher magnification of panel b. c, Liver sections from the indicated mice were stained with Oil red O for neutral lipids or with PAS for glycogen. The increased steatosis (Oil red O) and the effect on glycogen levels (PAS staining) in the STZ-treated Gly⁻ mice both corroborate the diabetes associated changes (low insulin and islet damage). Bar = 100 μm.

Figure 3. Immostaining of pancreata from K18 WT and Gly⁻ mice and biochemical assessment of apoptosis in liver and pancreas tissues

a, Pancreas sections were triple-stained for K8 (green), insulin (red) and nuclei (blue). Note that the K8 staining in Gly⁻pancreata after STZ treatment is lower because of severe edema, islet cell necrosis and blood cell infiltration. Arrows indicate infiltrating red blood cells in islet that express neither K8 nor insulin. Bar = 50 μm. b, c, Total lysates were prepared from mouse livers (b) and pancreata (c). The lysates were separated by SDS-PAGE then blotted with the indicated antibodies. A duplicate gel was stained with Coomassie blue to verify equal protein loading. Each lane represents the analysis of one independent organ.

Figure 4. K18 Gly⁻ predisposes transgenic mice to PUGNAc/Fas-induced injury

a, Nontransgenic FVB/n mice were injected intraperitoneally with the indicated doses of PUGNAc or vehicle (lane 1, PBS). Mice were then euthanized by CO₂ inhalation 48 hr after PUGNAc administration. Total liver lysates were prepared and immunoblotted with anti-O-GlcNAc antibody (Ab 110.6). An actin blot is shown as a loading control. b, c, FVB/n, K18 WT or K18 Gly⁻ mice were pretreated with PUGNAc (7 mg/kg body weight) for 48 hr to accumulate O-GlcNAc proteins, and then injected with Fas antibody (0.15 mg/kg mouse body weight). The mice were observed for 3 days (no deaths occurred after 3 days). Lethality (b) and survival curves (c) are shown. d, e, K18 WT or K18 Gly⁻mice were treated with PUGNAc ±Fas as in panels b and c. Livers were isolated 5 hr after Fas injection and used for the indicated stainings (d) and immunoblots (e). Bar in panel d = 200 μm.

Figure 5. K18 Gly⁻ alters protein kinase phosphorylation and promotes apoptosis in response to STZ

a, Hepatocytes were isolated from nontransgenic, K18-WT or K18-Gly⁻ mice and treated ex vivo with 5 mM STZ for the indicated times. Total cell lysates were then prepared, separated by SDS-PAGE, stained with Coomassie blue (to verify equal protein loading) and blotted with antibodies to the indicated antigens. b, K18 WT or Gly⁻ mice were injected i.p. with STZ or vehicle. Total liver lysates were prepared and analyzed as described in panel (a). Note that Akt1 T308 phosphorylation rose 12 hr after STZ in K18-WT livers (2.2 fold as determined by densitometric scanning) but remained flat up to 70 hr in K18-Gly⁻ livers. c, The phosphoinositide-dependent protein kinase 1 (PDK1) consensus motif of the catalytic domain of several protein kinases. The first Thr of the motif (shown in light font) is phosphorylated by PDK1. d, Total liver lysates similar to those used in panel b were blotted with antibodies to the indicated phospho and non-phospho PKC sites. PKCθ T538, PKCδ T505 and PKCξ/λ T410/402 are PDK1 substrates (panel c), whereas PKCθ/δ S676/643, PKCβII S660 and PKCα/βII T637/641 are not. e, Pancreas and liver lysates were isolated from K18 WT or Gly⁻ mice at the indicated times (+/− STZ administration), then analyzed by blotting with antibodies to the indicated antigens (actin is used as a loading control).

Figure 6. K18 Gly⁻ inhibits Akt T308 phosphorylation and Hsp70 expression, companying with enhanced apoptosis in response to PUNAc/Fas

a, b, FVB/n mice (a), or K18 WT and K18 Gly⁻ mice (b) were given PUGNAc (7 mg/kg body weight) or vehicle. Livers were harvested at the indicated times after PUGNAc injection and total liver lysates were blotted with antibodies to the indicated antigens. c, K18 WT or K18 Gly⁻ mice were pretreated with PUGNAc for 48 hr then injected with Fas Ab. Livers were harvested at 2, 5, 7, 9 hr after Fas injection and total liver lysates were immunoblotted with antibodies to the indicated antigens. Akt1 T308 phosphorylation rose 2 hr after PUGNAc+Fas in K18-WT livers (4.9 fold; compare lanes 1 and 2) but the increase in K18-Gly⁻ livers was limited. d, BHK cells were transfected with vector alone, Akt1 WT or T308A mutant. After 2 days, the transfected cells were treated with 100 μM PUGNAc for 18 hrs. Total cell lysates were prepared and blotted with antibodies to the indicated antigens. Note that O-GlcNAc proteins accumulated after PUGNAc treatment (lanes 4 and 5). The expression of Akt WT and T308A mutant were confirmed using Akt or phospho-specific Akt antibody. e, Transfected BHK cells (with Akt1 WT or T308A mutant) were treated with 100 μM PUGNAc for 18 hr. O-GlcNAc proteins were immunoprecipitated with two different O-GlcNAc antibodies, Ab110.6 or Ab RL2. The immunoprecipitates were analyzed by blotting with antibodies to Akt or vimentin (as a loading control). Note that similar patterns were obtained by using two different O-GlcNAc antibodies. The Akt1 T308A mutant was not efficiently immunoprecipitated with the O-GlcNAc antibodies as compared with Akt1 WT, which indicates that mutation of T308 inhibits Akt glycosylation and suggests that the O-GlcNAc modification occurs at or near Akt T308.

Figure 7. K8/K18 and Akt association is independent of PUGNAc/Fas treatment and Akt associates with keratin 8

a, BHK cells were transfected with the indicated cDNA constructs then treated with PUGNAc (100 μM, 18 hr). K8/K18 or Akt immunoprecipitates were obtained then analyzed by SDS-PAGE and Coomassie blue staining or were blotted with the indicated antibodies. b, K18 WT or K18 Gly⁻ mice were pretreated with PUGNAc (7 mg/kg body weight, 48 hr) and either injected with Fas antibody (0.15 mg/kg) or with saline (lanes 1,5). Livers were harvested 5, 7, 9 hr after Fas injection. K8/K18 and Akt immunoprecipitates were obtained then analyzed as in panel a. c, BHK cells were co-transfected with Akt WT and the indicated keratin constructs. K8 (1–253) and K18 (1–244) represent the N-terminal 253 or 244 amino acids, respectively. High salt extraction (HSE) was performed to generate a cytoskeletal preparation that contains primarily vimentin (the endogenous IF of BHK cells) and the transfected keratins followed by Coomassie blue staining (vimentin and K8 have similar migration under the analyzed conditions). The transfected cells undergo apoptosis due to the transfection stress conditions (Lipofectamine) and generate a stable 238 amino acid fragment as reported. Total cell lysates were also prepared from the transfected cells and blotted with antibody 8592 which recognizes K8 and K18. K8* and K18* represent major bands in the blot, while other weak bands in the same positions represent degraded K8 or background bands also found in Akt-transfectants (lane 1). Akt immunoprecipitates were also prepared and blotted with the indicated antibodies. d, BHK cells were transfected as in panel c. Akt immunoprecipitates were prepared and blotted with antibodies to K8/K18 or Akt. Expression of K8 WT and K8 (1–253) were similar as noted in the blot of the total lysates (panel c; lanes 3,4) (not shown). The displayed K8/K18 blot shows the details of the antibody reactive species in the range of 28–53 kd. Note that intact K8 WT (483 amino acids) co-precipitated with Akt (lane 2), but the K8 N-terminal fragment (1–253) did not (lane 3). The band marked with white arrow represents secondary antibody crossreactivity.

Figure 8. Model of K8 association with Akt and the effect of K18 glycosylation on reciprocal Akt phosphorylation and glycosylation

The schematic summarizes the results of the genetic animal models whereby ablation of K18 glycosylation or absence of the entire K18 protein in K18-null mice leads to Akt hypophosphorylation and inactivation which in turn promotes cell death. Inhibition of Akt T308 phosphorylation (by mutation to Akt T308A) inhibits Akt glycosylation (which may occur at T308 or an adjacent site), while hyperglycosylation of Akt (presumably at T308) leads to Akt hypophosphorylation at its regulatory T308.