Cell Cycle Markers Cyclin D1 Research Articles

Hepatic Deletion of X-Box Binding Protein 1 in FXR Null Mice Leads to Enhanced Liver Injury

FXR regulates bile acid metabolism, and FXR null (Fxr−/−) mice have elevated bile acid levels and progressive liver injury. The inositol-requiring enzyme 1α/X-box binding protein 1 (XBP1) pathway is a protective unfolded protein response pathway activated in response to endoplasmic reticulum stress. Here, we sought to determine the role of the inositol-requiring enzyme 1α/XBP1 pathway in hepatic bile acid toxicity using the Fxr−/− mouse model. Western blotting and quantitative PCR analysis demonstrated that hepatic XBP1 and other unfolded protein response pathways were activated in 24-week-old Fxr−/− compared with 10-week-old Fxr−/− mice but not in WT mice. To further determine the role of the liver XBP1 activation in older Fxr−/− mice, we generated mice with whole-body FXR and liver-specific XBP1 double KO (DKO, Fxr−/−Xbp1LKO) and Fxr−/−Xbp1fl/fl single KO (SKO) mice and characterized the role of hepatic XBP1 in cholestatic liver injury. Histologic staining demonstrated increased liver injury and fibrosis in DKO compared with SKO mice. RNA sequencing revealed increased gene expression in apoptosis, inflammation, and cell proliferation pathways in DKO mice. The proapoptotic C/EBP-homologous protein pathway and cell cycle marker cyclin D1 were also activated in DKO mice. Furthermore, we found that total hepatic bile acid levels were similar between the two genotypes. At age 60 weeks, all DKO mice and no SKO mice spontaneously developed liver tumors. In conclusion, the hepatic XBP1 pathway is activated in older Fxr−/− mice and has a protective role. The potential interaction between XBP1 and FXR signaling may be important in modulating the hepatocellular cholestatic stress responses.

LNX1 Contributes to Cell Cycle Progression and Cisplatin Resistance.

Simple SummaryThe ligand of numb-protein X1 (LNX1) is reported to be upregulated in various cancers, however the cellular function of LNX1 is not clearly characterized. The aim of the present study was to elucidate the regulation of LNX1 expression and clarify the role of LNX1 in cell-cycle progression and resistance to the cancer therapeutic agent, cisplatin. We found that LNX1 expression is decreased by DNA damage including cisplatin treatment and the levels of S and G2/M populations were correlated with LNX1 expression. We also showed that the upregulation of LNX1 contributes to cell-cycle progression and cisplatin resistance. Our data suggest that LNX1 is the important regulator of the cell cycle, and contributes to tumor progression.The ligand of numb-protein X1 (LNX1) acts as a proto-oncogene by inhibiting p53 stability; however, the regulation of LNX1 expression has not been investigated. In this study, we screened chemicals to identify factors that potentially regulate LNX1 expression. We found that LNX1 expression levels were decreased by DNA damage, including that by cisplatin. Upon treatment with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), LNX1 expression levels increased. In addition, cell-cycle progression increased upon LNX1 expression; the levels of S and G2/M populations were correlated with LNX1 expression. Moreover, in CRISPR-Cas9-mediated LNX1 knockout cells, we observed a delay in cell-cycle progression and a downregulation of genes encoding the cell-cycle markers cyclin D1 and cyclin E1. Finally, the upregulation of LNX1-activated cell-cycle progression and increased resistance to cisplatin-mediated cell death. Taken together, these results suggest that LNX1 contributes to cell-cycle progression and cisplatin resistance.

Clinico-pathological significance of Drp1 dysregulation and its correlation to apoptosis in oral cancer patients

Dysregulation in mitochondrial dynamics has been associated with several diseases including cancer. Present study assessed the alteration in mitochondrial fission protein (Drp1) in oral epithelial cells collected from clinically confirmed pre-cancer and cancer patients and further correlates it with the cellular apoptosis signaling. Results indicate the ROS accumulation in OSCC patients is accompanied by several changes including increase in mitochondrial mass, expression of mitochondrial fission protein (Drp1) and alteration in apoptotic signaling. The positive co-relation has been observed between the expressions of anti-apoptotic Bcl-2proteinswith mitochondrial fission protein Drp1. Higher mitochondrial fission in oral cancer cells was also correlated with the increased expression of cell cycle marker CyclinD1 indicating highly proliferative stage of oral cancer cells. The clinical correlation can be extended to develop biomarker for diagram and program in oral cancer management.

Roles of Id1/HIF-1 and CDK5/HIF-1 in cell cycle reentry induced by amyloid-beta peptide in post-mitotic cortical neuron

One neurotoxic mechanism of amyloid-beta peptide (Aβ), the major component of senile plaques in the brains of Alzheimer's disease (AD) patients, is to trigger cell cycle reentry in fully differentiated neurons. However, the detailed underlying mechanisms remain unclear. Using Aβ25-35–treated primary rat cortical neurons as the experimental system, in the present study we tested whether Aβ-induced inhibitor of differentiation-1 (Id1)/hypoxia-inducible factor-1alpha (HIF-1α) and cyclin-dependent kinase-5 (CDK5) contribute to cell cycle reentry in fully differentiated post-mitotic neurons. We found that Id1-induced HIF-1α mediated Aβ25-35–dependent expression of the cell cycle markers cyclin D1 and proliferating cell nuclear antigen (PCNA), both colocalized with microtubule-associated protein-2 (MAP-2) + cells, indicative of cell cycle reentry in the mature neurons. Aβ25-35 also enhanced p35 cleavage to p25 without affecting CDK5 expression. The CDK5 inhibitor roscovitine and the siRNA targeting CDK5 both suppressed Aβ25-35–dependent HIF-1α expression and cell cycle reentry. Intriguingly, Aβ25-35–induced Id1 repressed p25 production while CDK5/p25 reciprocally inhibited Id1 expression, despite the observation that both Id1 and CDK5/p25 acted upstream of HIF-1α. These results demonstrated that both Id1/HIF-1 and CDK5/HIF-1 contribute to Aβ-induced cell cycle reentry in post-mitotic neurons; furthermore, Id1 and CDK5/p25 reciprocally suppress expression of each other.

Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

BackgroundLong non-coding RNAs (lncRNAs) are tumor-associated biological molecules and have been found to be implicated in the progression of colorectal cancer (CRC). This study aims to examine the effects of lncRNA RP11-468E2.5 and its target genes (STAT5 and STAT6) on the biological activities of CRC cells via the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway.MethodsWe initially screened the GEO database for differentially expressed lncRNAs related to CRC and then made a prediction of the implicated target genes. Then we collected CRC tissues and adjacent normal tissues from 169 CRC patients. Human CRC HCT116 and SW480 cells were treated with small interference RNA (siRNA) against RP11-468E2.5, AG490 (an inhibitor of the JAK/STAT signaling pathway), or both in combination. Next, we measured the effects of RP11-468E2.5 treatment on cellular activities such as cell viability, cycle distribution and cell apoptosis, and studied interactions among RP11-468E2.5, STAT5/STAT6, and the JAK/STAT signaling pathway. Finally, an in vivo tumor formation assay was performed to observe the effect of RP11-468E2.5 on tumor growth.ResultsThe CRC-related gene microarray data showed low expression of RP11-468E2.5 in CRC surgical specimens. However, RP11-468E2.5 was confirmed to target STAT5 and STAT6, which participate in the JAK/STAT signaling pathway. CRC tissues showed lower expression of RP11-468E2.5, higher expression of STAT5, STAT6 and of the cell cycle marker Cyclin D1 (CCND1), compared to the findings in adjacent normal tissues. The treatment of siRNA against RP11-468E2.5 increased expression of JAK2, STAT3, STAT5, STAT6, CCND1 and Bcl-2 along with the extent of STAT3, STAT5 and STAT6 phosphorylation, while lowering expression of P21 and P27. Treatment with AG490 exhibited approximately opposite effects, whereas siRNA against RP11-468E2.5 treatment stimulated CRC cell proliferation and reduced cell apoptosis, while promoting cell cycle entry; AG490 treatment reversed these results.ConclusionsAltogether, we conclude that up-regulation of RP11-468E2.5 inhibits the JAK/STAT signaling pathway by targeting STAT5 and STAT6, thereby suppressing cell proliferation and promoting cell apoptosis in CRC.

Beyond its antioxidant properties: Quercetin targets multiple signalling pathways in hepatocellular carcinoma in rats

AimsHepatocellular carcinoma (HCC) pathogenesis involves the interplay of multiple signalling pathways. Notch and Hedgehog (Hh) are two major developmental pathways that act in concert to regulate adult cell repair. CK2α -serine-threonine kinase-down-regulation enhanced apoptotic activity and was proven beneficial for HCC patients. Quercetin is a bioactive flavonoid and has been shown to protect against HCC through its antioxidant activity. This study was carried out to elucidate the antineoplastic effect of quercetin through regulating both Notch and Hh pathways, apoptosis, cell proliferation and CK2α activity. Main methodsHepatocellular carcinoma was induced in male Sprague Dawley rats by thioacetamide. Quercetin was administered in both protective and curative doses. Parameters of liver function and oxidative stress were assessed. CK2α, Notch and Hh pathways were evaluated using RT-PCR and ELISA. Apoptosis was investigated by detecting caspase-3, caspase-8 and p53. Proliferative and cell cycle markers as cyclin D1 and Ki-67 were detected immunohistochemically. Key findingsQuercetin inhibited CK2α and downregulated mRNA and protein expression of Notch1 and Gli2. Quercetin also suppressed caspase-3 expression but not caspase-8. Quercetin elevated p53 expression whereas proliferative and cell cycle markers cyclin D1 and Ki-67 were downregulated. Markers of hepatic cellular integrity such as AST, ALT, ALP, GGT, albumin and bilirubin were significantly ameliorated. This was confirmed by histological examination. Quercetin also alleviated oxidative stress as shown by SOD, GSH, MDA and NO levels. SignificanceWe can conclude that in addition to its antioxidant power, quercetin blocked Notch, Hedgehog, regulated the apoptotic and proliferative pathways and inhibited CK2α in HCC.

Characterization of Corneal Epithelial Cells in Keratoconus.

PurposeWe studied the cellular characteristics of epithelial cells in the cone and extraconal periphery of corneas in keratoconus eyes.MethodsThis prospective observational study was conducted at Narayana Nethralaya Eye Institute. A total of 83 and 42 eyes with keratoconus and normal topography, respectively, were included in the study. Corneal epithelial cells were collected and analyzed for apoptosis, proliferation, epithelial-mesenchymal transition, and differentiation status using molecular and biochemical tools. Statistical analysis was performed using the Student's t-test.ResultsCorneal epithelial cells from the cone showed significantly higher expression of proapoptotic marker BAX (P < 0.005) compared to controls. Significantly elevated expression of cell cycle markers CYCLIN D1 (P < 0.005) and Ki67 (P < 0.005) were noted in the extraconal region compared to controls. Cells of the cone showed significantly higher ZO-1 (P < 0.005) and lower vimentin (P < 0.005) compared to controls. Significantly lower expression of the differentiation marker CK3/12 (P < 0.05) was observed in cones compared to controls.ConclusionsCones of keratoconic corneas show enhanced cell death, poor differentiation, proliferation and epithelial-mesenchymal transition. The cellular changes of the corneal epithelial cells in the cone and extraconal region differ significantly in a keratoconus corneas.Translational RelevanceCharacterization of patient-specific corneal epithelial cellular status in keratoconus has the potential to determine the optimal treatment and therapeutic outcomes paving the way towards personalized treatment in the future.

Critical role of β1 integrin in postnatal beta-cell function and expansion.

β1 integrin is essential for pancreatic beta-cell development and maintenance in rodents and humans. However, the effects of a temporal beta-cell specific β1 integrin knockout on adult islet function are unknown. We utilized a mouse insulin 1 promoter driven tamoxifen-inducible Cre-recombinase β1 integrin knockout mouse model (MIPβ1KO) to investigate β1 integrin function in adult pancreatic beta-cells. Adult male MIPβ1KO mice were significantly glucose intolerant due to impaired glucose-stimulated insulin secretion in vivo and ex vivo at 8 weeks post-tamoxifen. The expression of Insulin and Pancreatic and duodenal homeobox-1 mRNA was significantly reduced in MIPβ1KO islets, along with reductions in insulin exocytotic proteins. Morphological analyses demonstrated that beta-cell mass, islet density, and the number of large-sized islets was significantly reduced in male MIPβ1KO mice. Significant reductions in the phosphorylation of signaling molecules focal adhesion kinase, extracellular signal-regulated kinases 1 and 2, and v-Akt murine thymoma viral oncogene were observed in male MIPβ1KO islets when compared to controls. MIPβ1KO islets displayed a significant increase in protein levels of the apoptotic marker cleaved-Poly (ADP-ribose) polymerase and a reduction of the cell cycle marker cyclin D1. Female MIPβ1KO mice did not develop glucose intolerance or reduced beta-cell mass until 16 weeks post-tamoxifen. Glucose intolerance remained in both genders of aged MIPβ1KO mice. This data demonstrates that β1 integrin is required for the maintenance of glucose homeostasis through postnatal beta-cell function and expansion.

Scutellarin promotes microglia-mediated astrogliosis coupled with improved behavioral function in cerebral ischemia

Scutellarin, an anti-inflammatory agent, has been reported to suppress microglia activation. It promotes astrocytic reaction but through activated microglia. Here we sought to determine more specifically the outcomes of scutellarin treatment in reactive astrocytes in rats subjected to middle cerebral artery occlusion (MCAO). GFAP, MAP-2 and PSD-95 expression was assessed in reactive astrocytes in scutellarin injected MCAO rats. Expression of BDNF, NT-3 and IGF-1, and cell cycle markers cyclin-D1/B1 was also evaluated. In vitro, the above-mentioned proteins were also investigated in TNC 1 and primary astrocytes, treated respectively with conditioned medium from BV-2 microglia with or without pretreatment of scutellarin and lipopolysaccharide. Behavioral study was conducted to ascertain if scutellarin would improve the neurological functions of MCAO rats. In MCAO, reactive astrocytes in the penumbral areas were hypertrophic bearing long extending processes; expression of all the above-mentioned markers was markedly augmented. When compared to the controls, TNC1/primary astrocytes responded vigorously to conditioned medium derived from BV-2 microglia treated with scutellarin + lipopolysaccharide as shown by enhanced expression of all the above markers by Western and immunofluorescence analysis. By electron microscopy, hypertrophic TNC1 astrocytes in this group showed abundant microfilaments admixed with microtubules. In MCAO rats given scutellarin treatment, neurological scores were significantly improved coupled with a marked decrease in infarct size when compared with the matching controls. It is concluded that scutellarin is neuroprotective and that it can amplify astrogliosis but through activated microglia. Scutellarin facilitates tissue remodeling in MCAO that maybe linked to improvement of neurological functions.

Hemodynamic forces regulate developmental patterning of atrial conduction.

Anomalous action potential conduction through the atrial chambers of the heart can lead to severe cardiac arrhythmia. To date, however, little is known regarding the mechanisms that pattern proper atrial conduction during development. Here we demonstrate that atrial muscle functionally diversifies into at least two heterogeneous subtypes, thin-walled myocardium and rapidly conducting muscle bundles, during a developmental window just following cardiac looping. During this process, atrial muscle bundles become enriched for the fast conduction markers Cx40 and Nav1.5, similar to the precursors of the fast conduction Purkinje fiber network located within the trabeculae of the ventricles. In contrast to the ventricular trabeculae, however, atrial muscle bundles display an increased proliferation rate when compared to the surrounding myocardium. Interestingly, mechanical loading of the embryonic atrial muscle resulted in an induction of Cx40, Nav1.5 and the cell cycle marker Cyclin D1, while decreasing atrial pressure via in vivo ligation of the vitelline blood vessels results in decreased atrial conduction velocity. Taken together, these data establish a novel model for atrial conduction patterning, whereby hemodynamic stretch coordinately induces proliferation and fast conduction marker expression, which in turn promotes the formation of large diameter muscle bundles to serve as preferential routes of conduction.

Estrogens Act As a Dorsal-Ventral Limiting Factor To Pattern The Hematopoietic Stem Cell Niche

While the genetic control of hematopoietic stem cell (HSC) function is increasingly understood, less is known about factors that pattern the embryonic hematopoietic niche and specify the location of HSC emergence. 17beta-estradiol (E2) and related estrogenic compounds were identified in a zebrafish chemical genetic screen as modifiers of the number of runx1+ HSCs in the Aorta-Gonad-Mesonephros region. Exposure to exogenous E2 during hematopoietic specification significantly decreased production of runx1+ HSCs by in situ hybridization (ISH) and qPCR (p&lt;0.01); this finding was confirmed by CD41 FACS (p&lt;0.001). Morpholino knockdown of the estrogen receptors indicated E2-mediated loss of HSCs was due to Esr2a signaling. Zebrafish that express GFP under the control of the estrogen response element (ERE:GFP) showed endogenous estrogen activity and a 1.5 fold enhancement of GFP expression by FACS upon E2 treatment (p&lt;0.01) confirming that zebrafish embryos contain endogenous estrogen and the transcriptional machinery to respond to exogenous E2; estradiol immunoassay revealed that endogenous estrogen is preferentially located in the yolk of developing embryos. E2 treatment from 12-24 hours post fertilization (hpf), the time in which hemogenic endothelium is specified, disrupted vessel formation as shown by flk1 (kdrl) and altered the assignment of hemogenic arterial identity as assessed by the arterial markers ephrinB2a and tbx20. As arterial specification is mediated by a cascade of sonic hedgehog, VEGF, and Notch, we examined if these pathways were altered. Consistent with a defect in the specification of hemogenic endothelium, E2 decreased expression of VEGFAa and arterial Notch pathway components deltaC and notch3 by ISH and qPCR (p&lt;0.05). Induction of VEGFAa or Notch activity via heat-shock inducible lines rescued E2-mediated hematopoietic defects, suggesting that alterations in these networks underlie observed hematopoietic phenotypes. Further, repression of endogenous estrogen by the aromatase inhibitor anastrozole or the estrogen receptor antagonist fulvestrant increased levels of runx1, the arterial markers ephrinB2a and tbx20, and VEGFAa expression. Expression of venous flt4 was correspondingly decreased, demonstrating that endogenous estrogen signaling regulates arterial/venous identity. These results suggest that maternally deposited estrogens act as a novel morphogen organizer in patterning the HSC niche during early development. It further indicates that endogenous estrogen signaling acts to limit the ventral boundary of hemogenic endothelium by antagonizing somitic VEGFAa, thereby implicating estrogen as a critical regulator of the spatially restricted induction of HSCs. To determine if E2 could impact HSCs independently of its role in specification of hemogenic endothelium, we exposed embryos to E2 from 27-34 hpf, after arterial establishment and initiation of blood flow. Here, E2 enhanced HSCs by ISH and qPCR (runx1; p&lt;0.05). qPCR levels of the cell cycle markers cyclinD1 (p&lt;0.01) and c-myc (p&lt;0.001) and AGM phospho-histone H3 immunoreactivity (p&lt;0.01) increased, suggesting E2 enhances cell cycling. To determine if the ability of E2 to enhance proliferation was restricted to HSCs or was apparent on any blood progenitor population, we assessed the impact of E2 on primitive hematopoiesis. E2 treatment from 12-36 hpf enhanced expression of gata1 by ISH, FACS, and qPCR (p&lt;0.05) while repressing the expression of globin (p&lt;0.05), suggesting that while E2 enhances the self-renewal of progenitors, it impairs their differentiation into mature lineages. To analyze conservation of effect, we examined hematopoietic development in murine embryos with 5-alpha reductase deficiency. These embryos fail to metabolize testosterone into dihydroxytestosterone and as a result have more testosterone available for conversion by aromatase into estradiol, leading to increased estrogen levels. Homozygote pups show fewer phenotypic HSCs by FACS; heterozygotes display increased erythrocyte progenitors but no increase in mature erythrocytes, suggesting that the impact of estrogen on HSC induction and erythropoiesis is conserved in a mammalian system and is sensitive to dose. Together, these data indicate a novel role for estrogen in the regionalization of the hematopoietic niche and identify estrogen as an enhancer of HSC proliferation and maturation. Disclosures: No relevant conflicts of interest to declare.

Withania somnifera Water Extract as a Potential Candidate for Differentiation Based Therapy of Human Neuroblastomas

Neuroblastoma is an aggressive childhood disease of the sympathetic nervous system. Treatments are often ineffective and have serious side effects. Conventional therapy of neuroblastoma includes the differentiation agents. Unlike chemo-radiotherapy, differentiation therapy shows minimal side effects on normal cells, because normal non-malignant cells are already differentiated. Keeping in view the limited toxicity of Withania somnifera (Ashwagandha), the current study was aimed to investigate the efficacy of Ashwagandha water extract (ASH-WEX) for anti-proliferative potential in neuroblastoma and its underlying signalling mechanisms. ASH-WEX significantly reduced cell proliferation and induced cell differentiation as indicated by morphological changes and NF200 expression in human IMR-32 neuroblastoma cells. The induction of differentiation was accompanied by HSP70 and mortalin induction as well as pancytoplasmic translocation of the mortalin in ASH-WEX treated cells. Furthermore, the ASH-WEX treatment lead to induction of neural cell adhesion molecule (NCAM) expression and reduction in its polysialylation, thus elucidating its anti-migratory potential, which was also supported by downregulation of MMP 2 and 9 activity. ASH-WEX treatment led to cell cycle arrest at G0/G1 phase and increase in early apoptotic population. Modulation of cell cycle marker Cyclin D1, anti-apoptotic marker bcl-xl and Akt-P provide evidence that ASH-WEX may prove to be a promising phytotherapeutic intervention in neuroblatoma related malignancies.

Enhanced recovery of mechanical function in the canine heart by seeding an extracellular matrix patch with mesenchymal stem cells committed to a cardiac lineage

The need to regenerate tissue is paramount, especially for the heart that lacks the ability to regenerate after injury. The urinary bladder extracellular matrix (ECM), when used to repair a right ventricular defect, successfully regenerated some mechanical function. The objective of the current study was to determine whether the regenerative effect of ECM could be improved by seeding the patch with human mesenchymal stem cells (hMSCs) enhanced to differentiate down a cardiac linage. hMSCs were used to form three-dimensional spheroids. The expression of cardiac proteins was determined in cells exposed to the spheroid formation and compared with nonmanipulated hMSCs. To determine whether functional calcium channels were present, the cells were patch clamped. To evaluate the ability of these cells to regenerate mechanical function, the spheroids were seeded on ECM and then implanted into the canine heart to repair a full-thickness right ventricular defect. As a result, many of the cells spreading from the spheroids expressed cardiac-specific proteins, including sarcomeric alpha-actinin, cardiotin, and atrial natriuretic peptide, as well as the cell cycle markers cyclin D1 and proliferating cell nuclear antigen. A calcium current similar in amplitude to that of ventricular myocytes was present in 16% of the cells. The cardiogenic cell-seeded scaffolds increased the regional mechanical function in the canine heart compared with the unmanipulated hMSC-seeded scaffolds. In addition, the cells prelabeled with fluorescent markers demonstrated myocyte-specific actinin staining with sarcomere spacing similar to that of normal myocytes. In conclusion, the spheroid-derived cells express cardiac-specific proteins and demonstrate a calcium current similar to adult ventricular myocytes. When these cells are implanted into the canine heart, some of these cells appear striated and mechanical function is improved compared with the unmanipulated hMSCs. Further investigation will be required to determine whether the increased mechanical function is due to a differentiation of the cardiogenic cells to myocytes or to other effects.

The unfolded protein response affects neuronal cell cycle protein expression: Implications for Alzheimer's disease pathogenesis

Alzheimer's disease (AD) is characterized by the accumulation and aggregation of misfolded proteins. The presence of misfolded proteins in the endoplasmic reticulum (ER) triggers a cellular stress response called the unfolded protein response (UPR). Previously, we have shown that the UPR is activated in AD neurons. In actively dividing cells, activation of the UPR is accompanied by decreased cell cycle protein expression and an arrest in the G1 phase of the cell cycle. Aberrant expression of cell cycle proteins has been observed in post mitotic neurons in AD and is suggested to be involved in neurodegeneration. In this study we show that the protein levels of BiP/GRP78, an ER-stress marker, is increased in Braak stages B and C for amyloid deposits. This is in contrast to the levels of cell cycle markers cyclin D1, cyclin E and phosphorylated retinoblastoma protein (ppRb) which are decreased in Braak stage C compared to Braak stage A for amyloid deposits. In addition, we report a negative correlation between neuronal expression of ppRb and expression levels of BiP/GRP78 in control and AD cases. Activation of the UPR in neuronal cells induces changes in cell cycle protein expression similar to these observed in AD brain. ER stress inducers tunicamycin and thapsigargin down-regulate cell cycle proteins ppRb and cyclin D1 in differentiated neuroblastoma cells. In contrast, protein levels of p27, a cyclin dependent kinase inhibitor, are increased after induction of ER-stress using tunicamycin. These data suggest that activation of the UPR affects cell cycle protein expression in neurons during neurodegeneration in AD.

Nicotinamide induces apoptosis and reduces collagen I and pro-inflammatory cytokines expression in rat hepatic stellate cells

Nicotinamide has been shown to inhibit proliferation and induce apoptosis in a variety of cells. Moreover, nicotinamide treatment attenuates collagen accumulation and fibrogenesis in the bleomycin model of lung fibrosis. We hypothesized that nicotinamide may be useful as an antifibrotic agent in liver fibrosis and we investigated the in vitro effect of nicotinamide on hepatic stellate cells proliferation, apoptosis and collagen I expression. Transforming growth factor beta1 (TGF-beta1) was used for activation of the rat HSC-T6 cell line. Apoptosis was determined by fluorescence activated cell sorter (FACS) analysis after propidium iodide staining and by immunohistochemistry showing presence of the active form of caspase 3. Expression of activation marker alpha-smooth muscle actin (alpha-SMA), apoptotic and cell cycle markers cyclin D1, P53 and caspase 3 was determined by Western blotting. Collagen I expression was assessed by Northern blotting. Nicotinamide inhibits hepatic stellate cell proliferation and induces apoptosis with caspase-3 activation. There is no effect of nicotinamide on the levels of cell cycle stimulator cyclin D1. Expression of p53 is induced in the presence of nicotinamide. Nicotinamide reduces activation marker alpha-SMA and decreases both basal and TGFbetaepsilon-induced collagen I expression. Moreover, in TGFbeta-activated cells, nicotinamide reduces expression of pro-inflammatory and pro-fibrotic cytokines TGFbeta2, IL-1beta, TNFalpha and macrophage chemotactic protein-1. The in vitro effect of nicotinamide on activation and proliferation of hepatic stellate cells suggests that nicotinamide may have a potential beneficial role in attenuation of liver fibrogenesis.