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Mild cognitive impairment (MCI) is an early stage that can result in dementia. MCI can be reversed, and diagnosis at an early stage is crucial to control the progression to dementia. Dementia is currently diagnosed based on interviews and screening tests; however, novel biomarkers must be identified to allow early MCI detection. Therefore, the present study aimed to identify novel biomarkers in the form of blood microRNAs (miRNAs/miRs) for the diagnosis of MCI or early dementia. Blood samples were collected from C57BL/6NJcl male mice at four time points, including 4-week-old (4W), 8-week-old (8W), 36-week-old (36W) and 58-week-old (58W), and serum was isolated. Body weight and blood total cholesterol levels were increased, and blood alkaline phosphatase was decreased with aging. The 8W mice exhibited the highest cognitive ability in the Morris water maze test, whereas the 58W mice demonstrated decreased cognitive ability. The serum RNA concentrations of the 4W, 8W, 36W and 58W mice demonstrated no significant differences. Furthermore, small RNA levels were detected in the serum of all mice. miRNA microarray analysis revealed a >1.5-fold increase in the serum expression of two miRNAs (miR-21a-5p and miR-92a-3p) and a >1.5-fold decrease in the serum expression of two other miRNAs (miR-6769b-5p and miR-709) in 58W mice compared with those in 8W mice. In the future, we aim to further analyze aged mice to discover novel MCI biomarkers., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Yamamoto et al.)

Background and Aim: In chronic hepatic diseases where treatment strategies are not available, deposited fibrotic tissues deteriorate the intrinsic regeneration capacity of the liver by creating special restrictions. Thus, if the anti-fibrosis modality is efficiently applied, the regeneration capacity of the liver should be reactivated even in such refractory hepatic diseases., Methods: Rat liver fibrosis was induced by dimethyl-nitrosamine (DMN). Another liver fibrosis model was established in CCl4 treated Sox9CreERT2ROSA26: YFP mice. To resolve hepatic fibrosis, vitamin A-coupled liposomes containing siRNA HSP47 (VA-liposome siHSP47) were employed. EpCAM + hepatic progenitor cells from GFP rats were transplanted to DMN rat liver to examine their trans-differentiation into hepatic cells after resolution of liver fibrosis., Results: Even under continuous exposure to such strong hepatotoxin as DMN, rats undergoing VA-liposome siHSP47 treatment showed an increment of DNA synthesis of hepatocytes with the concomitant restoration of impaired liver weight and normalization of albumin levels. These results were consistent with the observation that GFP + EpCAM hepatic progenitor cells transplanted to DMN rat liver, trans-differentiated into GFP + mature hepatic cells after VA-liposome siHSP47 treatment. Another rodent model also proved regeneration potential of the fibrotic liver in CCl4 administered Sox9CreERT2ROSA26: YFP mice, VA-liposome siHSP47 treatment-induced restoration of liver weight and trans-differentiation of YEP + Sox9 + cells into YFP + hepatic cells, although because of relatively mild hepatotoxicity of CCl4, undamaged hepatocytes also proliferated., Conclusions: These results demonstrated that regeneration of chronically damaged liver indeed occurs after anti-fibrosis treatment even under continuous exposure to hepatotoxin, which promises a significant benefit of the anti-fibrosis therapy for refractory liver diseases., (© 2021 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Pancreatic cancer is among the most refractory malignancies with poor prognosis. Thus, preventive approaches, in addition to the development of novel therapeutic strategies are essential for this type of cancer. KRAS mutations occur very early in the development of pancreatic cancers and could be targeted for its prevention, yet specific inhibitors for mutated KRAS are lacking. Accordingly, Glutathione-S Transferase p1 (GSTP1), which we recently found to be an autocrine stimulator of mutated KRAS signaling, is predicted to be an alternative target for chemoprevention of pancreatic cancer. In this study, chemopreventive effects of O-Hexadecyl-γ-glutamyl-S-benzyl-cysteinyl-D-phenyl glycine-Ethylester (HGBPE), which we previously synthesized to inhibit GSTP1 activity, was analyzed for its effect on the prevention of a rat pancreatic carcinogenesis model induced by 7,12-dimethyl-benzanthracene (DMBA). Rats administered with DMBA were grouped into five cohorts. In the treated group I, which was treated neither with HGBPE nor vehicle, sequential appearance of precancerous lesions, ductal complexes, and adenocarcinoma was confirmed as previously reported. We also confirmed in this group that mutations of KRAS and expression of GSTP1 simultaneously occurred in the ductal complex. To rats of groups II and IV, HGBPE was administered, and vehicle to those of group III and V. In groups of II and IV, the incidence of both ductal complex and adenocarcinoma were significantly lower than those in groups III and V. These data clearly suggest the efficacy of HGBP as a potential chemopreventive agent for pancreatic cancer., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutant KRAS (m KRAS ) and BRAF (m BRAF ). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of m KRAS and m BRAF cancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of m KRAS and m BRAF cells in vitro and suppressed tumorigenesis of the xenografted m KRAS tumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of m KRAS colon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

CIP75 is a member of the UbL(ubiquitin-like)-UBA (ubiquitin-associated) domain containing protein family, which has a variety of functions. One specific role described for several members of the UbL-UBA family is the involvement in the proteasomal degradation of target proteins. We have reported that CIP75 interacts with the gap junction protein, connexin43 (Cx43), and that CIP75 may modulate the proteasomal degradation of Cx43. Thus, CIP75 may have a critical role in regulating Cx43 levels, and thus intercellular gap junctional communication. This study reports the development of monoclonal antibodies (MAbs) against CIP75 and the characterization of these antibodies through immunoblotting, immunoprecipitation, and immunofluorescence microscopy analyses. These MAbs will be useful tools in future studies to elucidate the role of CIP75 in Cx43 proteasomal degradation as well as other potential activities.

The mechanism by which v-Src disrupts connexin (Cx)43 intercellular gap junctional communication (GJC) is not clear. In this study, we determined that Tyr247 (Y247) and the previously identified Tyr265 (Y265) site of Cx43 were the primary phosphorylation targets for activated Src in vitro. We established an in vivo experimental system by stably expressing v-Src and wild-type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout mouse cell line. Wt and mutant Cx43 localized to the plasma membrane in the absence or presence of v-Src. When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. Most importantly, GJC established by the Y247F, Y265F, and Y247F/Y265F Cx43 mutants was resistant to disruption by v-Src. Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src. We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.

It is not clear how the v-Src oncoprotein disrupts gap junctional communication (GJC) established by connexin43 (Cx43) in mammalian cells. In this study, an experimental system was established to stably express v-Src and wild type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout (KO) mouse cell line. When co-expressed with v-Src, the levels of phosphotyrosine (pTyr) from Y247F, Y265F, and Y247F/Y265F Cx43 mutants were reduced to approximately 57%, 10%, and 2% of the level of pTyr from wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. These data also implied that phosphorylation of Cx43 at Y265 was required for efficient phosphorylation of Cx43 at Y247. Most importantly, our measurements of GJC demonstrated that, in contrast to the wt Cx43 gap junction channels, the Y247F, Y265F, and Y247F/Y265F Cx43 channels were resistant to the disruption by v-Src. In conclusion, our studies support a model for processive phosphorylation of Cx43 on tyrosine, at the Y265 site followed by the Y247 site, in mediating the disruption of GJC induced by v-Src in mammalian cells.

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.

The gap junction protein connexin43 is a phosphoprotein that typically migrates as three bands (nonphosphorylated, P1 and P2) during polyacrylamide gel electrophoresis. The electrophoretic mobility of connexin43 from mitotic cells was distinctly reduced to a form (P3) that migrated slower than P2 from Rat1 cells prepared by shakeoff of nocodazole-treated and untreated cultures. Mitotic FT210 cells, which contain a temperature-sensitive mutation in the p34(cdc2) kinase, showed abundant levels of the P3 connexin43 when maintained at the permissive temperature where p34(cdc2) is active. In contrast, nocodozole-treated FT210 cells grown at the nonpermissive temperature did not contain P3 connexin43. These results indicated that generation of the P3 connexin43 was dependent upon active p34(cdc2)/cyclin B kinase. Although the p34(cdc2)kinase phosphorylated connexin43 in vitro on peptides containing serine 255, the major phosphotryptic peptides in P3 connexin43 from mitotic cells appeared to be the consequence of another protein kinase(s), which may be activated by the p34(cdc2)/cyclin B kinase. The P3 connexin43 exhibited a marked redistribution from cell-cell plasma membrane interfaces to multiple, distinctly stained cytoplasmic structures. These events may be part of the dramatic structural changes observed in mitotic cells undergoing cell rounding and cytokinesis. Results of initial studies using inhibitors of protein degradative and synthetic pathways suggested the likelihood that protein degradation and synthesis participate in the disappearance of the P3 connexin43 and restoration of the pattern of connexin43 isoforms observed in nonmitotic cells.

Immortalized cells from embryonic connexin43 knockout mice (Cx43-/-) and homozygous littermates (Cx43+/+) were cloned and characterized to determine whether the absence of Cx43 function would induce observable phenotypic changes. Cells of the Cx43+/+ clones expressed Cx43 and engaged in gap junctional communication with 10-12 neighboring cells. The Cx43-/- cells were devoid of Cx43 and communicated to less than 1 cell. Electrophysiological analysis indicated that the Cx43-/- cells communicated through Cx45 channels from 8-80-fold less than did the Cx43+/+ subclones, which seemed to communicate through Cx43 and Cx45 channels. The Cx43-/- clones grew at faster rates and to higher saturation densities, had a more spindly morphology, were more refractile, and adhered less well to the substratum than did the Cx43+/+ clones. Reintroducing the Cx43 gene into the Cx43-/- clones resulted in three subclones that communicated to 3-4 cells. Partial restoration of gap junctional communication in the three subclones was accompanied by reduced growth rates and saturation densities (2-fold compared to that of parental Cx43-/- clones) but no reversions in morphology or cell-substratum adhesion. The increased growth rates and saturation densities, altered morphology, and decreased cell adhesion displayed by the Cx43-/- clones reflect a subset of the properties of transformed cells. These studies advance the hypothesis that loss of Cx43 function during development may cause cells to acquire a preneoplastic condition.

The lens gap-junction protein, connexin 56, is modified by phosphorylation. Two-dimensional mapping of tryptic phosphopeptides of 32P-labeled connexin 56 from primary chicken-lens cultures showed that treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) induced an increase in phosphorylation of connexin 56 at specific constitutively phosphorylated sites. Treatment with 8-Br-cAMP or forskolin did not induce substantial changes in connexin 56 phosphorylation. Two phosphorylation sites within connexin 56, S493 and S118, were identified after HPLC purification and peptide sequencing of tryptic phosphopeptides from bacterially expressed connexin 56 fusion proteins phosphorylated by protein kinase C or protein kinase A in vitro. Comparisons of the two-dimensional maps of tryptic phosphopeptides from in vitro phosphorylated connexin 56 fusion proteins and in vivo phosphorylated connexin 56 showed that S493 and S118 were constitutively phosphorylated in lentoid-containing cultures, and that treatment with TPA induced an increase in phosphorylation of the peptides containing S118. It is suggested that phosphorylation of connexin 56 at S118 is involved in the TPA-induced decrease in intercellular communication and acceleration of connexin 56 degradation.

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.

We have previously demonstrated that epidermal growth factor induced a rapid, transient decrease in gap junctional communication and increase in serine phosphorylation on the connexin-43 gap junction protein in T51B rat liver epithelial cells. The kinase(s) responsible for phosphorylation and specific serine targets in connexin-43 have not been identified. There are three consensus mitogen-activated protein (MAP) kinase serine phosphorylation sequences in the carboxyl-terminal tail of connexin-43 and purified MAP kinase phosphorylated connexin-43 in vitro on tryptic peptides that comigrated with a subset of peptides from connexin-43 phosphorylated in vivo in cells treated with epidermal growth factor. These data suggested that MAP kinase may phosphorylate connexin-43 directly in vivo. We have utilized a glutathione S-transferase fusion protein containing the cytoplasmic tail of connexin-43 to characterize MAP kinase phosphorylation. Site-directed mutagenesis, phosphotryptic peptide analysis, and peptide sequencing have confirmed that MAP kinase can phosphorylate connexin-43 at Ser255, Ser279, and Ser282, which correspond to the consensus sites recognized earlier. Characterization of MAP kinase-mediated phosphorylation of connexin-43 has defined potential targets for phosphorylation in vivo following activation of the epidermal growth factor receptor and has provided the basis for studies of the effects of phosphorylation, at specific molecular sites, on the regulation of gap junctional communication.

The pp60v-src tyrosine kinase disrupts gap junctional communication in transformed fibroblasts and induces the phosphorylation of the gap junction protein, connexin43, on tyrosine. We report here that the p130gag-fps tyrosine kinase also profoundly disrupted gap junctional communication and markedly increased the phosphorylation of connexin43 which appeared to result from an accumulation of phosphotyrosine and phosphoserine. The disruption of gap junctional communication by pp60v-src and p130gag-fps did not appear to result from the gross alteration of gap junction plaques. Furthermore, two-dimensional phosphotryptic peptide mapping showed that the v-Src and V-Fps kinases stimulated the phosphorylation of multiple connexin43 peptides which contained phosphotyrosine and/or phosphoserine. Phosphotyrosine was detected in two connexin43 phosphotryptic peptides from v-src-tranformed cells which suggested that more than one connexin43 tyrosine site may be recognized by pp60v-src in fibroblasts. The apparent higher levels of phosphoserine-containing connexin43 peptides in the oncogene-transformed cells pointed to the possibility that pp60v-src and p130gag-fps may also modulate connexin43 function through mechanism(s) involving the activation of signaling serine kinases. Taken together, these results suggested that connexin43 is a common target of the v-Src and v-Fps tyrosine kinase oncoproteins.

Growth factors regulate cellular proliferation and differentiation by activating plasma membrane tyrosine kinase receptors and triggering a cascade of events mediated by intracellular signaling proteins. The mechanism underlying growth factor modification of cellular functions, such as gap-junctional communication (gjc), has not been established clearly. Addition of epidermal growth factor (EGF) to T51B rat liver epithelial cells resulted in the rapid activation of EGF receptor tyrosine kinase activity followed by a transient dose-dependent disruption of gjc. This change did not result from the gross disturbance of membrane gap junction plaques as measured by immunofluorescence microscopy, but instead correlated with markedly elevated phosphorylation of the connexin43 (cx43) gap junction protein, a profound shift to predominantly phosphorylated forms of cx43, and the appearance of a novel phosphorylated cx43 protein. These changes in cx43 phosphorylation involved only serine residues. On restoration of gjc, these alterations in cx43 phosphorylation reverted to the pre-EGF treatment state. Both events were inhibited by the serine/threonine protein phosphatase inhibitor, okadaic acid. Therefore, unlike the case for pp60v-src, EGF-induced disruption of gjc is not associated with tyrosine phosphorylation of cx43, but instead may result from phosphorylation of cx43 by activated intracellular signaling serine protein kinase(s).

Disruption of gap junctional communication (measured by intercellular dye transfer) in cultured fibroblasts by pp60v-src is correlated with phosphorylation of the gap junction protein, connexin43 (cx43), on tyrosine. In this report, we examine the functional relevance of these observations by studying cx43 phosphorylation in cells containing kinase-active, non-myristylated pp60(2A527F) or pp60v-src temperature sensitive (ts) for transformation. Non-transformed cells expressing pp60(2A527F) transferred fluorescent dye at high levels and contained cx43 that was phosphorylated predominately on serine. In contrast, cells transformed by kinase-active, myristylated pp60(527F) did not transfer dye and contained cx43 proteins which were phosphorylated on serine and tyrosine. Additionally, activation of ts pp60v-src tyrosine kinase activity upon shift of cells to the permissive temperature was correlated with a rapid increase in the phosphorylated tyrosine content of cx43 proteins and loss of gap junctional communication. These combined results suggested that cx43 is a substrate of pp60v-src whose phosphorylation on tyrosine may be involved in the disruption of gap junctional communication observed in Rous sarcoma virus (RSV)-transformed cells.