DHEA and Its Metabolites Reduce the Cytokines Involved in the Inflammatory Response and Fibrosis in Primary Biliary Cholangitis.

Dehydroepiandrosterone (DHEA) is an abundant steroid and precursor of sex hormones. During aging, the reduction in DHEA synthesis causes a significant depletion of estrogens and androgens in different organs, such as the ovaries, brain, and liver. Primary Biliary Cholangitis (PBC) is a cholestatic liver disease that begins with immune-mediated bile duct damage, and is followed by liver fibrosis, and finally, cirrhosis. PBC primarily affects postmenopausal women, with an average age of diagnosis of 65 years, but younger women are also affected. Here, we analyzed the levels of DHEA, estradiol (E2), and estriol (E3) in the PBC sera of females at an age of diagnosis under 40 (n = 37) and above 65 (n = 29). Our results indicate that in PBC patients at an age of diagnosis under 40, E2 was significantly lower compared to that in healthy women. In contrast, the levels of DHEA and E3 were in a normal range. Furthermore, ELISA assays revealed that in PBC patients at an age of diagnosis above 65, the levels of DHEA, E2, and E3 significantly declined in comparison to those in younger patients. In addition, flow cytometry analysis showed that the level of IL-8 significantly decreased while the level of TNF-α increased in older PBC patients compared to younger ones. Moreover, we showed for the first time that the sulfonated form of DHEA, DHEA-S, reduces the levels of both pro-inflammatory interleukins, IL-8 and TNF-α, in PBC-like cholangiocytes (H69-miR506), while it diminishes the level of the pro-fibrotic interleukin, IL-13, in hepatocytes (Hep-G2). Finally, we demonstrated that the expression of the pro-fibrotic agent TGF-ß significantly increased in both the early (F0-F3) and cirrhotic (F4) stages of PBC, and this elevation was accompanied by higher α-SMA expression.

RNA-Seq Transcriptome Analysis of Differentiated Human Oligodendrocytic MO3.13 Cells Shows Upregulation of Genes Involved in Myogenesis.

In this work, we examined the differentiation of oligodendrocytic MO3.13 cells and changes in their gene expression after treatment with phorbol 12-myristate 13-acetate, PMA, or with RNA polymerase I (Pol I) inhibitor, CX-5461. We found that MO3.13 cells changed their morphology when treated with both agents. Interestingly, CX-5461, but not PMA, induced noticeable changes in the integrity of the nucleoli. Then, we analyzed the p53 transcriptional activity in MO3.13 cells and found that it was increased in both cell populations, but particularly in cells treated with PMA. Interestingly, this high p53 transcriptional activity in PMA-treated cells coincided with a lower level of an unmodified (non-phosphorylated) form of this protein. Since morphological changes in MO3.13 cells after PMA and CX-5461 treatment were evident, suggesting that cells were induced to differentiate, we performed RNA-seq analysis of PMA-treated cells, to reveal the direction of alterations in gene expression. The analysis showed that the largest group of upregulated genes consisted of those involved in myogenesis and K-RAS signaling, rather than those associated with oligodendrocyte lineage progression.

DHEA Protects Human Cholangiocytes and Hepatocytes against Apoptosis and Oxidative Stress.

Primary biliary cholangitis (PBC) is a rare chronic cholestatic and immune-mediated liver disease of unknown aetiology that targets intrahepatic bile duct cells (cholangiocytes) and primarily affects postmenopausal women, when their estrogen levels sharply decrease. An impaired cholangiocyte response to estrogen characterizes the terminal stage of the disease, as this is when an inefficiency of cholangiocyte proliferation, in balancing the loss of intrahepatic bile ducts, is observed. Here, we report that the estrogen precursor dehydroepiandrosterone (DHEA) and its sulfate metabolites, DHEA-S and 17 ß-estradiol, enhance the proliferation of cholangiocytes and hepatocytes in vitro. Flow cytometry analysis showed that DHEA and DHEA-S decreased glyco-chenodeoxycholic acid (GCDC)-driven apoptosis in cholangiocytes. Cell viability assay (MTT) indicated that ER-α, -ß, and the G-protein-coupled estrogen receptor, are involved in the protection of DHEA against oxidative stress in cholangiocytes. Finally, immunoblot analysis showed an elevated level of steroid sulfatase and a reduced level of sulfotransferase 1E1 enzymes, involved in the desulfation/sulfation process of estrogens in cirrhotic PBC, and primary sclerosis cholangitis (PSC) liver tissues, another type of chronic cholestatic and immune-mediated liver disease. Taken together, these results suggest that DHEA can prevent the deleterious effects of certain potentially toxic bile acids and reactive oxygen species, delaying the onset of liver disease.

p-STAT3 is a PDC-E2 interacting partner in human cholangiocytes and hepatocytes with potential pathobiological implications.

The E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC) is the key autoantigen in primary biliary cholangitis (PBC) and STAT3 is an inflammatory modulator that participates in the pathogenesis of many liver diseases. This study investigated whether PDC-E2 interacts with STAT3 in human cholangiocytes (NHC) and hepatocytes (Hep-G2) under cholestatic conditions induced by glyco-chenodeoxycholic acid (GCDC). GCDC induced PDC-E2 expression in the cytoplasmic and nuclear fraction of NHC, whereas in Hep-G2 cells PDC-E2 expression was induced only in the cytoplasmic fraction. GCDC-treatment stimulated phosphorylation of STAT3 in the cytoplasmic fraction of NHC. siRNA-mediated gene silencing of PDC-E2 reduced the expression of pY-STAT3 in NHC but not in HepG2 cells. Immunoprecipitation and a proximity ligation assay clearly demonstrated that GCDC enhanced pY-STAT3 binding to PDC-E2 in the nuclear and cytoplasmic fraction of NHC cells. Staining with Mitotracker revealed mitochondrial co-localization of PDC-E2/pS-STAT3 complexes in NHC and Hep-G2 cells. In cirrhotic PBC livers the higher expression of both PDC-E2 and pY-STAT3 was observed. The immunoblot analysis demonstrated the occurrence of double bands of PDC-E2 protein in control livers, which was associated with a lower expression of pY-STAT3. Our data indicate the interaction between PDC-E2 and phosphorylated STAT3 under cholestatic conditions, which may play a role in the development of PBC.

Pharmacological inhibition of spinal cord injury-stimulated ribosomal biogenesis does not affect locomotor outcome.

After unresolved endoplasmic reticulum stress, recovery of protein synthesis including increased expression of ribosomal components and translation factors may induce cell death. Using a mouse model of moderate contusive spinal cord injury (SCI) at the T9 level, upregulation of ribosomal biogenesis was observed in the injury epicenter at 24h after trauma. Such upregulation coincided with endoplasmic reticulum stress response as previously reported in this model. It was also accompanied by changes in expression of many other genes associated with translational regulation. Systemic treatment with a pharmacological inhibitor of RNA-Polymerase-1, BMH-21 reduced rRNA transcription in the spinal cord. Moreover, in the injury epicenter, treatment with BMH-21 increased expression of oligodendrocyte-specific transcripts including Mbp and Cldn11 at 3days post injury. Although such findings may suggest at least transient reduction of oligodendrocyte death, locomotor outcome was mostly unaffected except slightly accelerated recovery of hindlimb function at week 2 post-injury. Therefore, at least in mice, RNA-Polymerase-1 does not appear to be a robust target for therapies to protect spinal cord tissue after contusion. However, these findings raise an interesting possibility that altered rate of ribosomal biogenesis contributes to the apparent translational reprogramming after contusive SCI. Such a reprogramming could be a major regulator of SCI-induced gene expression.

Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione.

The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10(-8) M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter.

Influence of S100A6 on CacyBP/SIP Phosphorylation and Elk-1 Transcriptional Activity in Neuroblastoma NB2a Cells.

In this work, we have found that casein kinase II (CKII) phosphorylates the CacyBP/SIP protein under in vitro conditions and have mapped the phosphorylation site to threonine 184. Moreover, we present evidence that S100A6, a CacyBP/SIP interacting protein, inhibits this phosphorylation in the presence of Ca(2+). CacyBP/SIP phosphorylation by CKII was also observed in neuroblastoma NB2a cells. Interestingly, we have found that the effect of DRB, a CKII inhibitor, on CacyBP/SIP phosphorylation state is similar to that of S100A6 overexpression. Phosphorylation at threonine 184 seems to have an effect on CacyBP/SIP phosphatase activity since the T184E phosphorylation mimic mutant overexpressed in NB2a cells has lower phosphatase activity toward p-ERK1/2 when compared to the non-phosphorylable T184A mutant or to the wild-type protein. In conclusion, our data suggest that S100A6 and Ca(2+), through inhibiting CacyBP/SIP phosphorylation on threonine 184, are important regulators of CacyBP/SIP phosphatase activity and of ERK1/2-Elk-1 signaling pathway.

Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress.

CacyBP/SIP [calcyclin-binding protein/Siah-1 [seven in absentia homolog 1 (Siah E3 ubiquitin protein ligase 1)] interacting protein] is a multifunctional protein whose activity includes acting as an ERK1/2 phosphatase. We analyzed dimerization of mouse CacyBP/SIP in vitro and in mouse neuroblastoma cell line (NB2a) cells, as well as the structure of a full-length protein. Moreover, we searched for the CacyBP/SIP domain important for dimerization and dephosphorylation of ERK2, and we analyzed the role of dimerization in ERK1/2 signaling in NB2a cells. Cell-based assays showed that CacyBP/SIP forms a homodimer in NB2a cell lysate, and biophysical methods demonstrated that CacyBP/SIP forms a stable dimer in vitro. Data obtained using small-angle X-ray scattering supported a model in which CacyBP/SIP occupies an anti-parallel orientation mediated by the N-terminal dimerization domain. Site-directed mutagenesis established that the N-terminal domain is indispensable for full phosphatase activity of CacyBP/SIP. We also demonstrated that the oligomerization state of CacyBP/SIP as well as the level of post-translational modifications and subcellular distribution of CacyBP/SIP change after activation of the ERK1/2 pathway in NB2a cells due to oxidative stress. Together, our results suggest that dimerization is important for controlling phosphatase activity of CacyBP/SIP and for regulating the ERK1/2 signaling pathway.

Calcyclin-binding protein/Siah-1-interacting protein as a regulator of transcriptional responses in brain cells.

The calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP) is highly expressed in the brain and has been shown to regulate ß-catenin-driven transcription in thymocytes. Therefore, we investigated whether CacyBP/SIP plays a role as a transcriptional regulator in brain cells. In brain-derived neurotrophic factor (BDNF)- and forskolin-stimulated rat primary cortical neurons, overexpression of CacyBP/SIP enhanced transcriptional activity of the cAMP-response element (CRE). In addition, overexpressed CacyBP/SIP enhanced BDNF-mediated activation of the nuclear factor of activated T cells (NFAT) but not the serum response element (SRE). These stimulatory effects required an intact C-terminal domain of CacyBP/SIP. Moreover, in C6 rat glioma cells, the overexpressed CacyBP/SIP enhanced activation of CRE and NFAT following forskolin and serum stimulation, respectively. Conversely, knockdown of endogenous CacyBP/SIP reduced activation of CRE and NFAT but not of SRE. Taken together, these results indicate that CacyBP/SIP is a novel regulator of CRE- and NFAT-driven transcription.

Up-regulation of CacyBP/SIP during rat breast cancer development.

BACKGROUND: CacyBP/SIP (calcyclin binding protein/Siah-1 interacting protein) was originally discovered in Ehrlich ascities tumor cells but was later found also in many different tumors. METHODS: To better understand the function of CacyBP/SIP in carcinogenesis, we used immunohistochemistry, Western blotting, and RT-PCR assays to study the distribution and level of CacyBP/SIP in mammary tissues after tumor induction in rat with DMBA [(dimethylbenz[a]anthracene)]. Application of such a model allowed us to monitor changes in CacyBP/SIP level during development of breast cancer. RESULTS: We found that both the protein and mRNA levels of CacyBP/SIP gradually increased in pathologically changed tissues and were highest in tumors taken 8 weeks after DMBA treatment. Similar changes as for CacyBP/SIP were detected in the level of ß-catenin. CONCLUSION: Increase in CacyBP/SIP expression during development of breast cancer, observed early in the mammary tissues with only minimal pathological changes, might suggest an important role of this protein in the process of carcinogenesis.

S100A6 is transcriptionally regulated by ß-catenin and interacts with a novel target, lamin A/C, in colorectal cancer cells.

In this paper we document an increased expression of S100A6, a calcium binding protein of the S100 family, and its co-localization with ß-catenin in colorectal cancer tissues and in metastatic, SW620, versus non-metastatic, SW480, human colorectal cancer cell lines. Moreover, we show up-regulation of the S100A6 protein level in non-metastatic SW480 cells due to overexpression of ß-catenin as well as the activation of the S100A6 gene promoter upon cell transfection with ß-catenin and the TCF-Lef1 transcription factor. Since we found a high level of S100A6 in metastatic SW620 cells we searched for its interacting partners in the protein extract prepared from these cells. Using several methods we found that S100A6 interacts with lamin A/C, a protein known to be implicated in colon carcinogenesis. Our results reveal a novel and important network of relations and interactions between proteins potentially involved in colorectal cancer development and progression.

CacyBP/SIP phosphatase activity in neuroblastoma NB2a and colon cancer HCT116 cells.

Recently, we have reported that CacyBP/SIP could be a novel phosphatase for ERK1/2 kinase. In this work, we analyzed the CacyBP/SIP phosphatase activity toward ERK1/2 in 2 cell lines of different origin. We showed that overexpression of CacyBP/SIP in NB2a cells resulted in a lower level of phosphorylated ERK1/2 (P-ERK1/2) in the nuclear fraction while such overexpression in HCT116 cells had no effect on the level of P-ERK1/2. Moreover, we found that overexpression of CacyBP/SIP resulted in higher phosphatase activity in the nuclear fraction obtained from NB2a cells when compared with HCT116 cells. Using 2-D electrophoresis we showed that the pattern of spots representing CacyBP/SIP differed in these 2 cell lines and was probably due to a different phosphorylation state of this protein. We also established that after overexpression of CacyBP/SIP in NB2a cells, the amount of nuclear ß-catenin was low, while it remained high in HCT116 cells. Since NB2a cells have differentiation potential and HCT116 cells do not, our data suggest that different activity of CacyBP/SIP in these 2 cell lines might affect the ERK1/2 pathway in the differentiation or proliferation processes.

ERK1/2 is dephosphorylated by a novel phosphatase--CacyBP/SIP.

Recently, we have reported that the CacyBP/SIP protein binds ERK1/2 (Kilanczyk et al., BBRC, 2009). In this work we show that CacyBP/SIP exhibits a phosphatase activity toward ERK1/2 kinases while its E217K mutant does not. The K(m) and V(max) values established for a standard phosphatase substrate, p-NPP, are 16.9±3.6 mM and 4.3±0.4 µmol/min, respectively. The CacyBP/SIP phosphatase activity is decreased by okadaic acid (IC(50)=45 nM). Our experimental results are supported by a theoretical analysis which revealed important sequence similarities between CacyBP/SIP and the phosphatase-like proteins as well as certain MAP kinase phosphatases.

CacyBP/SIP binds ERK1/2 and affects transcriptional activity of Elk-1.

In this work we showed for the first time that mouse CacyBP/SIP interacts with extracellular signal regulated kinases 1 and 2 (ERK1/2). We also established that a calcium binding protein, S100A6, competes for this interaction. Moreover, the E217K mutant of CacyBP/SIP does not bind significantly to ERK1/2 although it retains the ability to interact with S100A6. Molecular modeling shows that the E217K mutation in the 189-219 CacyBP/SIP fragment markedly changes its electrostatic potential, suggesting that the binding with ERK1/2 might have an electrostatic character. We also demonstrate that CacyBP/SIP-ERK1/2 interaction inhibits phosphorylation of the Elk-1 transcription factor in vitro and in the nuclear fraction of NB2a cells. Altogether, our data suggest that the binding of CacyBP/SIP with ERK1/2 might regulate Elk-1 phosphorylation/transcriptional activity and that S100A6 might further modulate this effect via Ca(2+)-dependent interaction with CacyBP/SIP and competition with ERK1/2.

CacyBP/SIP interacts with tubulin in neuroblastoma NB2a cells and induces formation of globular tubulin assemblies.

CacyBP/SIP, originally identified as a S100A6 (calcyclin) target, was later shown to interact with some other members of the S100 family as well as with Siah-1 and Skp1 proteins. Recently, it has been shown that CacyBP/SIP is up-regulated during differentiation of cardiomyocytes. In this work we show that the level of CacyBP/SIP is higher in differentiated neuroblastoma NB2a cells than in undifferentiated ones and that in cells overexpressing CacyBP/SIP the level of GAP-43, a marker of differentiation, was increased. Since the process of differentiation is accompanied by an extensive rearrangement of microtubules, we examined whether CacyBP/SIP interacted with tubulin. By applying cross-linking experiments we found that these two proteins bind directly. The dissociation constant of the tubulin-CacyBP/SIP complex determined by the surface plasmon resonance technique is 1.57 x 10(-7 )M which suggests that the interaction is tight. The interaction and co-localization of CacyBP/SIP and tubulin was also demonstrated by co-immunoprecipitation, affinity chromatography and immunofluorescence methods. Light scattering measurements and electron microscopy studies revealed that CacyBP/SIP, but not its homologue, Sgt1, increased tubulin oligomerization. Altogether, our results suggest that CacyBP/SIP, via its interaction with tubulin, might contribute to the differentiation of neuroblastoma NB2a cells.

The effect of melatonin on antioxidant enzymes in human diabetic skin fibroblasts.

Melatonin plays several important physiological functions in mammals, such as immune enhancement and regulation of dark-light signal transduction. Melatonin is also known to be an endogenous free radical scavenger and an efficient antioxidant. It detoxifies a variety of free radicals and reactive oxygen intermediates, including the hydroxyl radical, singlet oxygen and nitric oxide. These radicals participate in many diseases, for example diabetes. This study determined the effect of melatonin on the antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and the level of glutathione (GSH) in human diabetic (C2 line) skin fibroblasts. Confluent monolayers of control (S2 line) and diabetic (C2 line) skin fibroblasts were incubated with different concentrations of melatonin: 10, 50, 100 and 1000 micromol/l at 37 degrees C for 24 h. Next, the GSH level and SOD, CAT and GPx activities were measured colorimetrically. The activities of the antioxidant enzymes and the GSH level were lower in diabetic skin fibroblasts than in the control S2 line. Concentrations of melatonin of 100 and 1000 micromol/l caused a significant increase in the enzymes' activities and GSH level.

Effect of red laser light on Na+,K(+)-ATPase activity in human erythrocyte membranes sensitized with Zn-phthalocyanine.

OBJECTIVE: The influence of laser light (670 nm) on human erythrocyte membrane Na+,K(+)-ATPase activity in the presence and absence of Zn-phthalocyanine (ZnPc) was studied. BACKGROUND DATA: The response of erythrocyte membranes to low-power laser irradiation has not been fully elucidated. In our study, we focused on the studies on photo-induced changes of Na+,K(+)-ATPase activity. The erythrocyte membrane suspensions were incubated with 2 mM of ZnPc and next irradiated with energy doses of 19.1, 38.2, 57.3, 76.4, and 95.5 J x cm(-2). MATERIALS AND METHODS: The activity of Na+,K(+)-ATPase was assayed colorimetrically at the wavelength of 820 nm and expressed in micromol of inorganic phosphate released from ATP per mg of protein. RESULTS: The measurements of Na+,K(+)-ATPase activity in erythrocyte membranes incubated with ZnPc in the dark demonstrated that all concentrations of the dye (0.5, 1, 2, and 3 microM) stimulated enzyme activity. The concentration of 2 microM caused the smallest increase of enzyme activity, so this concentration was accepted for further studies. Irradiation of erythrocyte membranes in the presence of the dye (2 microM) significantly decreased Na+,K(+)-ATPase activity. Only for energy doses of 19.1 and 38.2 J x cm(-2) was the enzyme activity comparable to the activity of the control. CONCLUSION: It was found that irradiation with all energy doses applied caused a rise of enzyme activity. In the presence of ZnPc, significant decrease of Na+,K(+)-ATPase activity was observed.