Redundant roles of AKT1 and AKT2 in fertility, estrous cyclicity and endometrial gland development.

In brief: The regulation of AKT in the endometrium during many cellular processes such as apoptosis and cell survival is crucial during the estrous cycle to ensure fertility. This research shows the specific function of AKT isoforms in the mouse endometrium for litter size, estrous cyclicity and endometrial gland development. Abstract: Apoptosis and cell survival regulation are crucial processes during the estrous cycle to prepare a receptive uterus during implantation for successful recognition of pregnancy. PI3K/AKT signaling has a crucial role during gestation, and AKT isoforms (1, 2 or 3) are regulated differently in the endometrium during the estrous cycle and embryo implantation. However, the specific roles of these isoforms are still unclear. We have previously shown that AKT isoforms expression during the rat estrous cycle and gestation is differently regulated. The present study aimed to establish the specific role of AKT isoforms in the mouse uterus. The hypothesis is that dysregulation of AKT isoforms expression could cause fertility-related issues in an isoform-specific manner. With four different mouse models and in-house crossbreeding, all isoforms KO combinations (single, double and triple) were obtained in progesterone receptor-expressing tissues. The results demonstrated that in absence of one or more AKT isoforms, female fertility was decreased. Mainly, we have observed smaller litter size, specifically in Akt1-2 KO mice. Additionally, we have found Akt1-2-3 KO mice to be fully infertile. Estrous cyclicity was also disrupted in Akt1-2 KO mice with longer diestrus stage. Moreover, the number of endometrial glands was decreased throughout the estrous cycle suggesting an important role in gland development for AKT1 and AKT2. Our results suggest not only specific roles between each isoform but also a partially redundant function of AKT1 and AKT2 in litter size, estrous cyclicity and endometrial gland development. This highlights the importance of AKT in the physiological regulation of mouse fertility.

Pharmacologic inhibition of Akt in combination with chemotherapeutic agents effectively induces apoptosis in ovarian and endometrial cancer cell lines.

The PI3K/Akt signaling pathway, the most frequently altered signaling system in human cancer, is a crucial inducer of dysregulated proliferation and neoplastic processes; however, few therapeutic strategies using PI3K/Akt inhibitors singly have been shown to be effective. The purpose of this paper was to underline the potential benefit of pharmacological modulation of the PI3K/Akt pathway when combined with specific chemotherapeutic regimens. We have studied the ability of NVP-BEZ235 (PI3K/mTOR inhibitor) and AZD5363 (Akt inhibitor) in the sensitization of cancer cells to cisplatin and doxorubicin. Our results show that NVP-BEZ235 sensitizes cells preferentially to cisplatin while AZD5363 sensitizes cells to doxorubicin. At equal concentrations (5 µm), both inhibitors reduce ribosomal protein S6 phosphorylation, but AZD5363 is more effective in reducing GSK3ß phosphorylation as well as S6 phosphorylation. Additionally, AZD5363 is capable of inducing FOXO1 and p53 nuclear localization and reduces BAD phosphorylation, which is generally increased by cisplatin and doxorubicin. Finally, the combination of AZD5363 and doxorubicin induces apoptosis in cells and robustly reduces cell ability to clonally replicate, which underlines a potential cooperative effect of the studied compounds.

Keratin 8 and 18 loss in epithelial cancer cells increases collective cell migration and cisplatin sensitivity through claudin1 up-regulation.

Keratins 8 and 18 (K8/18) are simple epithelial cell-specific intermediate filament proteins. Keratins are essential for tissue integrity and are involved in intracellular signaling pathways that regulate cell response to injuries, cell growth, and death. K8/18 expression is maintained during tumorigenesis; hence, they are used as a diagnostic marker in tumor pathology. In recent years, studies have provided evidence that keratins should be considered not only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression plays an active role in EMT. We show that K8/18 stable knockdown using shRNA increased collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells showed PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increased cisplatin-induced apoptosis. Increased Fas receptor membrane targeting suggests that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and supports the hypothesis that keratins play an active role in cancer progression.

Functional specificity of Akt isoforms in cancer progression.

Akt/PKB kinases are central mediators of cell homeostasis. There are three highly homologous Akt isoforms, Akt1/PKBα, Akt2/PKBß and Akt3/PKBγ. Hyperactivation of Akt signaling is a key node in the progression of a variety of human cancer, by modulating tumor growth, chemoresistance and cancer cell migration, invasion and metastasis. It is now clear that, to understand the mechanisms on how Akt affects specific cancer cells, it is necessary to consider the relative importance of each of the three Akt isoforms in the altered cells. Akt1 is involved in tumor growth, cancer cell invasion and chemoresistance and is the predominant altered isoform found in various carcinomas. Akt2 is related to cancer cell invasion, metastasis and survival more than tumor induction. Most of the Akt2 alterations are observed in breast, ovarian, pancreatic and colorectal carcinomas. As Akt3 expression is limited to some tissues, its implication in tumor growth and resistance to drugs mostly occurs in melanomas, gliomas and some breast carcinomas. To explain how Akt isoforms can play different or even opposed roles, three mechanisms have been proposed: tissue-specificity expression/activation of Akt isoforms, distinct effect on same substrate as well as specific localization through the cyto-skeleton network. It is becoming clear that to develop an effective anticancer Akt inhibitor drug, it is necessary to target the specific Akt isoform which promotes the progression of the specific tumor.

Novel insights into changes in biochemical properties of keratins 8 and 18 in griseofulvin-induced toxic liver injury.

Keratins 8 and 18 (K8/18) intermediate filament proteins are believed to play an essential role in the protection of hepatocytes against mechanical and toxic stress. This assertion is mainly based on increased hepatocyte fragility observed in transgenic mice deficient in K8/18, or carrying mutations on K8/18. The molecular mechanism by which keratins accomplish their protective functions has not been totally elucidated. Liver diseases such as alcoholic hepatitis and copper metabolism diseases are associated with modifications, in hepatocytes, of intermediate filament organisation and the formation of K8/18 containing aggregates named Mallory-Denk bodies. Treatment of mice with a diet containing griseofulvin induces the formation of Mallory-Denk bodies in hepatocytes. This provides a reliable animal model for assessing the molecular mechanism by which keratins accomplish their protective role in the response of hepatocytes to chemical injuries. In this study, we found that griseofulvin intoxication induced changes in keratin solubility and that there was a 5% to 25% increase in the relative amounts of soluble keratin. Keratin phosphorylation on specific sites (K8 pS79, K8 pS436 and K18 pS33) was increased and prominent in the insoluble protein fractions. Since at least six K8 phosphoepitopes were detected after GF treatment, phosphorylation sites other than the ones studied need to be accounted for. Immunofluorescence staining showed that K8 pS79 epitope was present in clusters of hepatocytes that surrounded apoptotic cells. Activated p38 MAPK was associated with, but not present in K8 pS79-positive cells. These results indicate that griseofulvin intoxication mediates changes in the physicochemical properties of keratin, which result in the remodelling of keratin intermediate filaments which in turn could modulate the signalling pathways in which they are involved by modifying their binding to signalling proteins.

Akt isoforms regulate intermediate filament protein levels in epithelial carcinoma cells.

Keratin 8 and 18 are simple epithelial intermediate filament (IF) proteins, whose expression is differentiation- and tissue-specific, and is maintained during tumorigenesis. Vimentin IF is often co-expressed with keratins in cancer cells. Recently, IF have been proposed to be involved in signaling pathways regulating cell growth, death and motility. The PI3K/Akt pathway plays a pivotal role in these processes. Thus, we investigated the role of Akt (1 and 2) in regulating IF expression in different epithelial cancer cell lines. Over-expression of Akt1 increases K8/18 proteins. Akt2 up-regulates K18 and vimentin expression by an increased mRNA stability. To our knowledge, these results represent the first indication that Akt isoforms regulate IF expression and support the hypothesis that IFs are involved in PI3K/Akt pathway.

From Mallory to Mallory-Denk bodies: what, how and why?

Frank B. Mallory described cytoplasmic hyaline inclusions in hepatocytes of patients with alcoholic hepatitis in 1911. These inclusions became known as Mallory bodies (MBs) and have since been associated with a variety of other liver diseases including non-alcoholic fatty liver disease. Helmut Denk and colleagues described the first animal model of MBs in 1975 that involves feeding mice griseofulvin. Since then, mouse models have been instrumental in helping understand the pathogenesis of MBs. Given the tremendous contributions made by Denk to the field, we propose renaming MBs as Mallory-Denk bodies (MDBs). The major constituents of MDBs include keratins 8 and 18 (K8/18), ubiquitin, and p62. The relevant proteins and cellular processes that contribute to MDB formation and accumulation include the type of chronic stress, the extent of stress-induced protein misfolding and consequent proteasome overload, a K8-greater-than-K18 ratio, transamidation of K8 and other proteins, presence of p62 and autophagy. Although it remains unclear whether MDBs serve a bystander, protective or injury promoting function, they do serve an important role as histological and potential progression markers in several liver diseases.

Keratin 20 serine 13 phosphorylation is a stress and intestinal goblet cell marker.

Keratin polypeptide 20 (K20) is an intermediate filament protein with preferential expression in epithelia of the stomach, intestine, uterus, and bladder and in Merkel cells of the skin. K20 expression is used as a marker to distinguish metastatic tumor origin, but nothing is known regarding its regulation and function. We studied K20 phosphorylation as a first step toward understanding its physiologic role. K20 phosphorylation occurs preferentially on serine, with a high stoichiometry as compared with keratin polypeptides 18 and 19. Mass spectrometry analysis predicted that either K20 Ser(13) or Ser(14) was a likely phosphorylation site, and Ser(13) was confirmed as the phospho-moiety using mutation and transfection analysis and generation of an anti-K20-phospho-Ser(13) antibody. K20 Ser(13) phosphorylation increases after protein kinase C activation, and Ser(13)-to-Ala mutation interferes with keratin filament reorganization in transfected cells. In physiological contexts, K20 degradation and associated Ser(13) hyperphosphorylation occur during apoptosis, and chemically induced mouse colitis also promotes Ser(13) phosphorylation. Among mouse small intestinal enterocytes, K20 Ser(13) is preferentially phosphorylated in goblet cells and undergoes dramatic hyperphosphorylation after starvation and mucin secretion. Therefore, K20 Ser(13) is a highly dynamic protein kinase C-related phosphorylation site that is induced during apoptosis and tissue injury. K20 Ser(13) phosphorylation also serves as a unique marker of small intestinal goblet cells.

Heat shock protein 70 expression, keratin phosphorylation and Mallory body formation in hepatocytes from griseofulvin-intoxicated mice.

BACKGROUND: Keratins are members of the intermediate filaments (IFs) proteins, which constitute one of the three major cytoskeletal protein families. In hepatocytes, keratin 8 and 18 (K8/18) are believed to play a protective role against mechanical and toxic stress. Post-translational modifications such as phosphorylation and glycosylation are thought to modulate K8/18 functions. Treatment of mouse with a diet containing griseofulvin (GF) induces, in hepatocytes, modifications in organization, expression and phosphorylation of K8/18 IFs and leads, on the long term, to the formation of K8/18 containing aggregates morphologically and biochemically identical to Mallory bodies present in a number of human liver diseases. The aim of the present study was to investigate the relationship between the level and localization of the stress inducible heat shock protein 70 kDa (HSP70i) and the level and localization of K8/18 phosphorylation in the liver of GF-intoxicated mice. The role of these processes in Mallory body formation was studied, too. The experiment was carried out parallely on two different mouse strains, C3H and FVB/n. RESULTS: GF-treatment induced an increase in HSP70i expression and K8 phosphorylation on serines 79 (K8 S79), 436 (K8 S436), and K18 phosphorylation on serine 33 (K18 S33) as determined by Western blotting. Using immunofluorescence staining, we showed that after treatment, HSP70i was present in all hepatocytes. However, phosphorylated K8 S79 (K8 pS79) and K8 S436 (K8 pS436) were observed only in groups of hepatocytes or in isolated hepatocytes. K18 pS33 was increased in all hepatocytes. HSP70i colocalized with MBs containing phosphorylated K8/18. Phophorylation of K8 S79 was observed in C3H mice MBs but was not present in FVB/n MBs. CONCLUSIONS: Our results indicate that GF intoxication represents a stress condition affecting all hepatocytes, whereas induction of K8/18 phosphorylation is not occurring in every hepatocyte. We conclude that, in vivo, there is no direct relationship between GF-induced stress and K8/18 phosphorylation on the studied sites. The K8/18 phosphorylation pattern indicates that different cell signaling pathways are activated in subpopulations of hepatocytes. Moreover, our results demonstrate that, in distinct genetic backgrounds, the induction of K8/18 phosphorylation can be different.