The intermediate filament protein keratin 8 (K8) is critical for the development of most mouse embryos beyond midgestation. We find that 68% of K8-/- embryos, in a sensitive genetic background, are rescued from placental bleeding and subsequent death by cellular complementation with wild-type tetraploid extraembryonic cells. This indicates that the primary defect responsible for K8-/- lethality is trophoblast giant cell layer failure. Furthermore, the genetic absence of maternal but not paternal TNF doubles the number of viable K8-/- embryos. Finally, we show that K8-/- concepti are more sensitive to a TNF-dependent epithelial apoptosis induced by the administration of concanavalin A (ConA) to pregnant mothers. The ConA-induced failure of the trophoblast giant cell barrier results in hematoma formation between the trophoblast giant cell layer and the embryonic yolk sac in a phenocopy of dying K8-deficient concepti in a sensitive genetic background. We conclude the lethality of K8-/- embryos is due to a TNF-sensitive failure of trophoblast giant cell barrier function. The keratin-dependent protection of trophoblast giant cells from a maternal TNF-dependent apoptotic challenge may be a key function of simple epithelial keratins.

Keratin 8 and keratin 18 (K8/K18) are intermediate filament proteins that form the obligate heteropolymers in hepatocytes and protect the liver against toxins. The mechanisms of protection include the regulation of signaling pathway associated with cell survival. Previous studies show K8/K18 binding with Akt, which is a well-known protein kinase involved in the cell survival signaling pathway. However, the role of K8/K18 in the Akt signaling pathway is unclear. In this study, we found that K8/K18-Akt binding is downregulated by K8/K18 phosphorylation, specifically phosphorylation of K18 ser7/34/53 residues, whereas the binding is upregulated by K8 gly-62-cys mutation. K8/K18 expression in cultured cell system tends to enhance the stability of the Akt protein. A comparison of the Akt signaling pathway in a mouse system with liver damage shows that the pathway is downregulated in K18-null mice compared with nontransgenic mice. K18-null mice with Fas-induced liver damage show enhanced apoptosis combined with the downregulation of the Akt signaling pathway, i.e., lower phosphorylation levels of GSK3β and NFκB, which are the downstream signaling factors in the Akt signaling pathway, in K18-null mice compared with the control mice. Our study indicates that K8/K18 expression protects mice from liver damage by participating in enhancing the Akt signaling pathway.

Keratins 8 and 18 (K8/18) are major constituents of Mallory bodies (MBs), which are hepatocyte cytoplasmic inclusions seen in several liver diseases. K18-null but not K8-null or heterozygous mice form MBs, which indicates that K8 is important for MB formation. Early stages in MB genesis include K8/18 hyperphosphorylation and overexpression. We used transgenic mice that overexpress K8, K18, or K8/18 to test the importance of K8 and/or K18 in MB formation. MBs were induced by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Livers of young K8 or K8/K18 overexpressors had no histological abnormalities despite increased keratin protein and phosphorylation. In aging mice, only K8-overexpressing livers spontaneously developed small "pre-MB" aggregates. Only K8-overexpressing young mice are highly susceptible to MB formation after short-term DDC feeding. Thus, the K8 to K18 ratio, rather than K8/18 overexpression by itself, plays an essential role in MB formation. K8 overexpression is sufficient to form pre-MB and primes animals to accumulate MBs upon DDC challenge, which may help explain MB formation in human liver diseases.

Monoclonal antibodies that specifically recognize caspase cleaved K18 fragments or specific (phospho)epitopes on intact K8 and K18 were used for a detailed investigation of the temporal and causal relationship of proteolysis and phosphorylation in the collapse of the keratin cytoskeleton during apoptosis. Caspases involved in the specific proteolysis of keratins were analyzed biochemically using recombinant caspases and specific caspase inhibitors. Finally, the fate of the keratin aggregates was analyzed using the M30-ApoptoSense trade mark Elisa kit to measure shedding of caspase cleaved fragments into the supernatant of apoptotic cell cultures. From our studies, we conclude that C-terminal K18 cleavage at the (393)DALD/S site is an early event during apoptosis for which caspase 9 is responsible, both directly and indirectly by activating downstream caspases 3 and 7. Cleavage of the L1-2 linker region of the central alpha-helical rod domain is responsible for the final collapse of the keratin scaffold into large aggregates. Phosphorylation facilitates formation of these aggregates, but is not crucial. K8 and K18 remain associated in heteropolymeric aggregates during apoptosis. At later stages of the apoptotic process, that is, when the integrity of the cytoplasmic membrane becomes compromised, keratin aggregates are shed from the cells.

Here, we report on the analysis of keratin 18 null mice. Unlike the ablation of K8, which together with K18 is expressed in embryonic and simple adult epithelia, K18 null mice are viable, fertile, and show a normal lifespan. In young K18 null mice, hepatocytes were completely devoid of keratin filaments. Nevertheless, typical desmosomes were formed and maintained. Old K18 null mice, however, developed a distinctive liver pathology with abnormal hepatocytes containing K8-positive aggregates. These stained positively for ubiquitin and MM120-1 and were identified as Mallory bodies, one hallmark of human alcoholic hepatitis. This is the first demonstration that the ablation of one keratin leads to the accumulation of its single partner. Another striking finding was the absence or drastic down regulation of K7 in several tissues despite its ongoing transcription. Moreover, K18 null mice revealed new insights in the filament-forming capacity of the tail-less K19 in vivo. Due to the unexpected secondary loss of K7, only K8/19 are expressed in the uterine epithelium of K18 null mice. Immunoelectron microscopy of this tissue demonstrated the presence of typical K8/19 IF, thus highlighting in vivo that K19 is a fully competent partner for K8.

Keratins, the intermediate filament proteins of epithelial cells, connect to desmosomes, the cell-cell adhesion structures at the surface membrane. The building elements of desmosomes include desmoglein and desmocollin, which provide the actual cell adhesive properties, and desmoplakins, which anchor the keratin intermediate filaments to desmosomes. In the work reported here, we address the role of keratin 8 in modulating desmoplakin deposition at surface membrane in mouse hepatocytes. The experimental approach is based on the use of keratin 8- and keratin 18-null mouse hepatocytes as cell models. In wild-type mouse hepatocytes, desmoplakin is aligned with desmoglein and keratin 8 at the surface membrane. In keratin 8-null hepatocytes, the intermediate filament loss leads to alterations in desmoplakin distribution at the surface membrane, but not of desmoglein. Intriguingly, a significant proportion of keratin 18-null hepatocytes express keratin 8 at the surface membrane, associated with a proper desmoplakin alignment with desmoglein at desmosomes. A Triton treatment of the monolayer reveals that most of the desmoplakin present in either wild-type, keratin 8- or keratin 18-null hepatocytes is insoluble. Deletion analysis of keratin 8 further suggests that the recovery of desmoplakin alignment requires the keratin 8 rod domain. In addition, similarly to other works revealing a key role of desmoplakin phosphorylation on its interaction with intermediate filaments, we find that the phosphorylation status of the keratin 8 head domain affects desmoplakin distribution at desmosomes. Together, the data indicate that a proper alignment/deposition of desmoplakin with keratins and desmoglein in hepatocytes requires keratin 8, through a reciprocal phosphoserine-dependent process.

Steatohepatitis (SH) and SH-associated hepatocellular carcinoma (HCC) are of considerable clinical significance. SH is morphologically characterized by steatosis, liver cell ballooning, cytoplasmic aggregates termed Mallory-Denk bodies (MDBs), inflammation, and fibrosis at late stage. Disturbance of the keratin cytoskeleton and aggregation of keratins (KRTs) are essential for MDB formation.

Toll-like receptors (TLRs) have critical roles in innate immunity and inflammation and the detailed mechanisms by which TLR signaling is fine tuned remain unclear. Keratin 8 (CK8) belongs to the type II keratin family and is the major compontent of the intermediate filaments of simple or single-layered epithelia. Here we report that down-regulation of CK8 in mice enhanced TLR-mediated responses, rendering mice more susceptible to lipopolysaccharide (LPS)-induced endotoxin shock and Escherichia coli-caused septic peritonitis with reduced survival, elevated levels of inflammation cytokines and more severe tissue damage. We found that CK8 suppressed TLR-induced nuclear factor (NF)-κB activation and interacted with the adaptor tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to prevent its polyubiquitination. Our findings demonstrate a novel role of CK8 in negative regulation of TLR/NF-κB signaling and highlight a previously unidentified nonclassical function for CK8 in limiting inflammatory responses.

Diabetes is a result of an interplay between genetic, environmental and lifestyle factors. Keratin intermediate filaments are stress proteins in epithelial cells, and keratin mutations predispose to several human diseases. However, the involvement of keratins in diabetes is not well known. K8 and its partner K18 are the main β-cell keratins, and knockout of K8 (K8-/- ) in mice causes mislocalization of glucose transporter 2, mitochondrial defects, reduced insulin content and altered systemic glucose/insulin control. We hypothesize that K8/K18 offer protection during β-cell stress and that decreased K8 levels contribute to diabetes susceptibility.

Resistin is associated with metabolic syndrome and inflammatory conditions. Many studies have suggested that resistin inhibits the accumulation of glycogen; however, the exact mechanisms of resistin-induced decrease in glycogen content remain unclear. Keratin 8 is a typical epithelial intermediate filament protein, but numerous studies suggest a vital role of K8 in glucose metabolism. However, it is still not known whether K8 participates in the mediation of resistin-induced reduction of cellular glycogen accumulation. In this study, we found that resistin upregulated expression of the p65 subunit of NF-κB, which led to the promotion of K8 transcriptional expression; in turn, the expression of K8 inhibited glycogen accumulation in HepG2 cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the highest-grade malignancies in the world. More effective biomarkers and treatment plans are necessary to improve the diagnosis rate and clinical outcome. The oncogenesis of PDAC is influenced by several factors, including chronic pancreatitis (CP). Keratin 8 (KRT8) is an important member of the keratin protein family and plays a role in regulating the cellular response to stress stimuli and mediating inflammatory reactions. However, the role of KRT8 in pancreatitis and PDAC is still poorly understood. Here we assessed the differentially expressed genes (DEGs) by bioinformatic methods with expression profiles available online for a caerulein-induced mouse model and human PDAC tissue. The prognostic value was evaluated by Kaplan-Meier analysis and Cox regression analysis. The diagnostic value was evaluated by Receiver Operating Characteristic analysis (ROC). The function of the genes was predicted by protein-protein interaction analysis, correlation analysis, and GO analysis. The conclusion was further validated in rat pancreatitis model, human tissue, and PDAC cell lines, including immunohistochemical staining (IHC), CCK-8 assay, wound healing assay, and flow cytometry. KRT8 was found to be upregulated in murine pancreatitis tissue, human CP tissue, and human PDAC tissue. High expression of KRT8 had a negative impact on the prognosis of PDAC patients. KRT8 was predicted to be involved in the regulation of the migration and viability of PDAC cells, which was validated in PDAC cell lines. Knockdown of KRT8 impaired the migration and proliferation and induced apoptosis in PDAC cell lines. In conclusion, keratin 8 is an inflammation-induced molecule and could serve as a diagnostic and prognostic marker for PDAC patients. More studies are needed for further validation from the perspective of precision and individualized medicine.

Keratins (Ks), the intermediate filament (IF) proteins of epithelia, are coordinately expressed as pairs in a cell-lineage and differentiation manner. Cortical thymic epithelial cells (cTECs) predominantly express the simple epithelium keratin 8/18 (K8/K18) pair, whereas medullary thymic epithelial cells (mTECs) express the stratified epithelium K5/K14 pair, with TECs exhibiting K5 and K8 at the cortico-medullary junction in mature thymus. In the work reported here, we used wild-type (WT) and K8-knockout (K8-null) mice to address the contribution of K8/K18 IFs in the maintenance of the thymic epithelial structure. K8-null thymus maintained the differential cell segregation at the cortex versus the medulla observed in WT thymus, and the distribution of immature thymocytes at the cortex. The K8/K18 loss did not affect thymocyte development. However, it massively perturbed the TEC morphology both at the cortex and the medulla, along with a prominent depletion of cTECs. Such tissue alterations coincided with an increase in apoptosis and a reduced expression of Albatross (Fas-binding factor-1), also known for its capacity to bind K8/18 IFs. In addition, the K8/K18 loss affected the distribution of K5/K14-positive mTECs, but not their differentiation status. Together, the results indicate that K8/K18 IFs constitute key promoters of the thymic epithelium integrity.

Lysine acetylation is an important posttranslational modification that regulates microtubules and microfilaments, but its effects on intermediate filament proteins (IFs) are unknown. We investigated the regulation of keratin 8 (K8), a type II simple epithelial IF, by lysine acetylation. K8 was basally acetylated and the highly conserved Lys-207 was a major acetylation site. K8 acetylation regulated filament organization and decreased keratin solubility. Acetylation of K8 was rapidly responsive to changes in glucose levels and was up-regulated in response to nicotinamide adenine dinucleotide (NAD) depletion and in diabetic mouse and human livers. The NAD-dependent deacetylase sirtuin 2 (SIRT2) associated with and deacetylated K8. Pharmacologic or genetic inhibition of SIRT2 decreased K8 solubility and affected filament organization. Inhibition of K8 Lys-207 acetylation resulted in site-specific phosphorylation changes of K8. Therefore, K8 acetylation at Lys-207, a highly conserved residue among type II keratins and other IFs, is up-regulated upon hyperglycemia and down-regulated by SIRT2. Keratin acetylation provides a new mechanism to regulate keratin filaments, possibly via modulating keratin phosphorylation.

Simple-type epithelial keratins are intermediate filament proteins important for mechanical stability and stress protection. Keratin mutations predispose to human liver disorders, whereas their roles in intestinal diseases are unclear. Absence of keratin 8 (K8) in mice leads to colitis, decreased Na/Cl uptake, protein mistargeting, and longer crypts, suggesting that keratins contribute to intestinal homeostasis. We describe the rate-limiting enzyme of the ketogenic energy metabolism pathway, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), as a major down-regulated protein in the K8-knockout (K8(-/-)) colon. K8 absence leads to decreased quantity and activity of HMGCS2, and the down-regulation is not dependent on the inflammatory state, since HMGCS2 is not decreased in dextran sulfate sodium-induced colitis. Peroxisome proliferator-activated receptor α, a transcriptional activator of HMGCS2, is similarly down-regulated. Ketogenic conditions-starvation or ketogenic diet-increase K8(+/+) HMGCS2, whereas this response is blunted in the K8(-/-) colon. Microbiota-produced short-chain fatty acids (SCFAs), substrates in the colonic ketone body pathway, are increased in stool, which correlates with decreased levels of their main transporter, monocarboxylate transporter 1 (MCT1). Microbial populations, including the main SCFA-butyrate producers in the colon, were not altered in the K8(-/-). In summary, the regulation of the SCFA-MCT1-HMGCS2 axis is disrupted in K8(-/-) colonocytes, suggesting a role for keratins in colonocyte energy metabolism and homeostasis.

Sphingosylphosphorylcholine (SPC) is significantly increased in the malicious ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments in PANC-1 cells. The reorganization contributes to the viscoelasticity of metastatic cancer cells resulting in increased migration. Recently, we reported that transglutaminase-2 (Tgase-2) is involved in SPC-induced K8 phosphorylation and reorganization. However, effects of Tgase-2 inhibitors on SPC-induced K8 phosphorylation and reorganization were not clearly studied. We found that ethacrynic acid (ECA) concentration-dependently inhibited Tgase-2. Therefore, we examined the effects of ECA on SPC-induced K8 phosphorylation and reorganization. ECA concentration-dependently suppressed the SPC-induced phosphorylation and perinuclear reorganization of K8. ECA also suppressed the SPC-induced migration and invasion. SPC induced JNK activation through Tgase-2 expression and ECA suppressed the activation and expression of JNK in PANC-1 cells. These results suggested that ECA might be useful to control Tgase-2 dependent metastasis of cancer cells such as pancreatic cancer and lung cancers.

Keratin 8 (K8) is a major intermediate filament protein present in enterocytes and serves an antiapoptotic function in hepatocytes. K8-null mice develop colonic hyperplasia and colitis that are reversed after antibiotic treatment. To investigate the pathways that underlie the mechanism of colonocyte hyperplasia and the normalization of the colonic phenotype in response to antibiotics, we performed genome-wide microarray analysis. Functional annotation of genes that are differentially regulated in K8(-/-) and K8(+/+) isolated colon crypts (colonocytes) identified apoptosis as a major altered pathway. Exposure of K8(-/-) colonocytes or colon organ ("organoid") cultures, but not K8(-/-) small intestine organoid cultures, to apoptotic stimuli showed, surprisingly, that they are resistant to apoptosis compared with their wild-type counterparts. This resistance is not related to inflammation per se because T-cell receptor α-null (TCR-α(-/-)) and wild-type colon cultures respond similarly upon induction of apoptosis. Following antibiotic treatment, K8(-/-) colonocytes and organ cultures become less resistant to apoptosis and respond similarly to the wild-type colonocytes. Antibiotics also normalize most differentially up-regulated genes, including survivin and β4-integrin. Treatment of K8(-/-) mice with anti-β4-integrin antibody up-regulated survivin, and induced phosphorylation of focal adhesion kinase with decreased activation of caspases. Therefore, unlike the proapoptotic effect of K8 mutation or absence in hepatocytes, lack of K8 confers resistance to colonocyte apoptosis in a microflora-dependent manner.

Specific biomarkers of intestinal injury associated with necrotizing enterocolitis (NEC) are needed to diagnose and monitor intestinal mucosal injury and recovery. This study aims to develop and test a modified enzyme-linked immunosorbent assay (ELISA) protocol to detect the total keratin 8 (K8) in the stool of newborns with NEC and investigate the clinical value of fecal K8 as a marker of intestinal injury specifically associated with NEC. We collected fecal samples from five newborns with NEC and five gestational age-matched premature neonates without NEC at the Lucile Packard Children's Hospital Stanford and Washington University School of Medicine, respectively. Fecal K8 levels were measured using a modified ELISA protocol and Western blot, and fecal calprotectin was measured using a commercial ELISA kit. Clinical data, including gestational age, birth weight, Bell stage for NEC, feeding strategies, total white blood cell (WBC) count, and other pertinent clinical variables, were collected and analyzed. Fecal K8 levels were significantly higher in the pre-NEC group (1-2 days before diagnosis of NEC) and NEC group than those in the non-NEC group (p = 0.013, p = 0.041). Moreover, fecal K8 was relatively higher at the onset of NEC and declined after the resolution of the disease (p = 0.019). Results with similar trends to fecal K8 were also seen in fecal calprotectin (p = 0.046), but not seen in total WBC count (p = 0.182). In conclusion, a modified ELISA protocol for the total K8 protein was successfully developed for the detection of fecal K8 in the clinical setting of premature newborns with NEC. Fecal K8 is noted to be significantly increased in premature newborns with NEC and may, therefore, serve as a noninvasive and specific marker for intestinal epithelial injury associated with NEC.

The stiffness of cancer cells is attributable to intermediate filaments such as keratin. Perinuclear reorganization via phosphorylation of specific serine residue in keratin is implicated in the deformability of metastatic cancer cells including the human pancreatic carcinoma cell line (PANC-1). 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter and protein kinase C (PKC) activator. However, its effects on phosphorylation and reorganization of keratin 8 (K8) are not well known. Therefore, we examined the underlying mechanism and effect of TPA on K8 phosphorylation and reorganization. TPA induced phosphorylation and reorganization of K8 and transglutaminase-2 (Tgase-2) expression in a time- and dose-dependent manner in PANC-1 cells. These effects peaked after 45 min and 100 nM of TPA treatment. We next investigated, using cystamine (CTM), Tgase inhibitor, and Tgase-2 gene silencing, Tgase-2's possible involvement in TPA-induced K8 phosphorylation and reorganization. We found that Tgase-2 gene silencing inhibited K8 phosphorylation and reorganization in PANC-1 cells. Tgase-2 gene silencing, we additionally discovered, suppressed TPA-induced migration of PANC-1 cells and Tgase-2 overexpression induced migration of PANC-1 cells. Overall, these results suggested that TPA induced K8 phosphorylation and reorganization via Tgase-2 expression in PANC-1 cells.

Hyperphosphorylated keratin (K) 8 acts as a phosphate "sponge" for stress-activated protein kinases thereby inhibiting pro-apoptotic molecules and thus apoptosis. MAP kinase/ERK1 has increased activity in colorectal cancer (CRC) and is known to phosphorylate K8. The aims were to identify the K8 isoforms abundantly present in colon tumors, using 2D difference gel electrophoresis (DIGE), to identify the modifications using mass spectrometry, and to validate the differential abundance of these isoforms in tumors relative to matched normal mucosae. 2D DIGE showed 3 isoforms of K8 significantly increased in tumor ≥2-fold in 6/8 pairs. Metal oxide affinity chromatography mass spectrometry and bioinformatics were used to identify phosphorylated serine residues. Levels of PS24, PS432, and PS74 by western blotting were found to be significantly increased in tumor versus matched normal. Blocking of EGFR signaling in Caco2 cells showed a significant decrease (P < 0.0001) in K8 PS74 and PS432 levels by 59% and 66%, respectively, resulting in increased apoptosis.

Inflammation is an important risk factor in atherosclerosis, the underlying cause of coronary artery disease (CAD). Unresolved inflammation may result in maladaptive immune responses and lead to immune reactivity to self-antigens. We hypothesized that inflammation in CAD patients would manifest in immune reactivity to self-antigens detectable in soluble HLA-I/peptide complexes in the plasma.