Keratin 18 is an integral part of the intermediate filament network in murine skeletal muscle.

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18-knockout (Krt18-KO) or dominant-negative mutant mice (Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18-null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.

Identification of morphological and biochemical changes in keratin-8/18 knock-down cells using Raman spectroscopy.

Accurate understanding of cellular processes and responses to stimuli is of paramount importance in biomedical research and diagnosis. Raman spectroscopy (RS), a label-free and nondestructive spectroscopic method has the potential to serve as a novel 'theranostics' tool. Both fiber-optic and micro-Raman studies have demonstrated efficacy in diagnostics and therapeutic response monitoring. In the present study, we have evaluated the potential of micro-Raman spectroscopic maps in identifying changes induced by loss of K8/18 proteins in a tongue cancer cell line. Furthermore, we also evaluated the efficacy of less expensive and commercially available fiber probes to identify K8/18 wild and knock-down cell pellets, in view of the utility of cell pellet-based studies. The findings suggest that major differences in the cellular morphology and biochemical composition can be objectively identified and can be utilized for classification using both micro-Raman and fiber-probe-based RS. These findings highlight the potential of fiber-optic probe-based RS in noninvasive cellular phenotyping for diagnosis and therapeutic response monitoring, especially in low-resource settings.

Keratin 18-deficiency results in steatohepatitis and liver tumors in old mice: A model of steatohepatitis-associated liver carcinogenesis.

Backround: Steatohepatitis (SH)-associated liver carcinogenesis is an increasingly important issue in clinical medicine. SH is morphologically characterized by steatosis, hepatocyte injury, ballooning, hepatocytic cytoplasmic inclusions termed Mallory-Denk bodies (MDBs), inflammation and fibrosis. RESULTS: 17-20-months-old Krt18-/- and Krt18+/- mice in contrast to wt mice spontaneously developed liver lesions closely resembling the morphological spectrum of human SH as well as liver tumors. The pathologic alterations were more pronounced in Krt18-/- than in Krt18+/- mice. The frequency of liver tumors with male predominance was significantly higher in Krt18-/- compared to age-matched Krt18+/- and wt mice. Krt18-deficient tumors in contrast to wt animals displayed SH features and often pleomorphic morphology. aCGH analysis of tumors revealed chromosomal aberrations in Krt18-/- liver tumors, affecting loci of oncogenes and tumor suppressor genes. MATERIALS AND METHODS: Livers of 3-, 6-, 12- and 17-20-months-old aged wild type (wt), Krt18+/- and Krt18-/- (129P2/OlaHsd background) mice were analyzed by light and immunofluorescence microscopy as well as immunohistochemistry. Liver tumors arising in aged mice were analyzed by array comparative genomic hybridization (aCGH). CONCLUSIONS: Our findings show that K18 deficiency of hepatocytes leads to steatosis, increasing with age, and finally to SH. K18 deficiency and age promote liver tumor development in mice, frequently on the basis of chromosomal instability, resembling human HCC with stemness features.

Loss of keratins 8 and 18 leads to alterations in α6ß4-integrin-mediated signalling and decreased neoplastic progression in an oral-tumour-derived cell line.

Keratins 8 and 18 (K8 and K18) are predominantly expressed in simple epithelial tissues and perform both mechanical and regulatory functions. Aberrant expression of K8 and K18 is associated with neoplastic progression and invasion in squamous cell carcinomas (SCCs). To understand the molecular basis by which K8 promotes neoplastic progression in oral SCC (OSCC), K8 expression was inhibited in AW13516 cells. The K8-knockdown clones showed a significant reduction in tumorigenic potential, which was accompanied by a reduction in cell motility, cell invasion, decreased fascin levels, alterations in the organization of the actin cytoskeleton and changes in cell shape. Furthermore, K8 knockdown led to a decrease in α6ß4 integrin levels and α6ß4-integrin-dependent signalling events, which have been reported to play an important role in neoplastic progression in epithelial tissues. Therefore, modulation of α6ß4 integrin signalling might be one of the mechanisms by which K8 and K18 promote malignant transformation and/or progression in OSCCs.

The prognostic potential of keratin 18 in breast cancer associated with tumor dedifferentiation, and the loss of estrogen and progesterone receptors.

The differential expression profiling with breast normal and tumor tissues, and a breast cancer cell line led to identification of cytokeratin 18 (KT18) gene over-expressed in breast cancer. The expression pattern of KT18 in breast cancer was compared to those of conventional tumor markers such as proliferating cell nuclear antigen (PCNA) and minichromosome maintenance protein 3 (MCM3). Their expression patterns in breast cancer were almost identical, suggesting that KT18 might be useful for detection of proliferating fractions in the breast cancer. The immunohistochemical analyses on the tissue microarray consisting of invasive ductal carcinomas revealed that the up-regulation of KT18 is observed in a majority of breast carcinomas whereas its down-regulation also occurs at a less frequency. Of particular interest, KT18 down-regulation was associated with histologically poorly differentiated carcinomas than well differentiated carcinomas. In addition, it was also significantly associated with the loss of estrogen receptor (ER) and progesterone receptor (PR), the prognostic markers of the breast cancer (P < 0.05), while not with HER2, tumor size, and lymph node metastasis. This result suggests that KT18 correlated with ER and PR may be utilized for the prognosis of breast cancer. Furthermore, the forced down-regulation of KT18 enhanced the growth of tumor xenografts in vivo and invasiveness in vitro. Therefore, our findings suggest that loss of KT18 expression might be a good indicator of the poor prognosis of the breast cancer and it may play an active role in the breast tumorigenesis.

The Pirh2-keratin 8/18 interaction modulates the cellular distribution of mitochondria and UV-induced apoptosis.

Intermediate filaments (IFs) provide crucial structural support in higher eukaryotic cells. Accumulating evidences show that IFs also participate in various cellular activities including stress responses, cell growth, cell death and cell migration through dynamic interactions with various non-structure proteins. Here we report the identification of Pirh2, a RING-H2-type ubiquitin E3 ligase, as a novel binding partner of the cytoplasmic IF proteins keratin 8/18 (K8/18). Phosphorylation of either Pirh2 or K8/18 affects their association. Although Pirh2 was not found to influence the stability of K8/18, it displayed an unexpected role in regulating the organization of the network of K8/18 keratin filaments. Disruption of Pirh2-K8/K18 interaction by either UV irradiation or knockdown with Pirh2 or K18 led to the aggregation of K8/18 keratin filaments. It further induced mitochondrial redistribution, and this process is likely through a microtubule-mediated pathway. The abnormal localization of mitochondria in Pirh2-knockdown cells may partially account for its increased cell sensitivity to UV-induced apoptosis, probably through enhancing the release of pro-apoptotic proteins, such as cytochrome c and Smac/DIABLO to the cytosol. Overall, our data reveal the novel role of the Pirh2-K8/18 complex in governing the distribution of mitochondria.