Salivary gland epithelial cell in Sjögren's syndrome: Metabolic shift and altered mitochondrial morphology toward an innate immune cell function.

Salivary gland epithelial cells (SGEC) are the main targets of the autoimmune reactivity in Sjögren's syndrome (SS). This study aimed to investigate the core proteomic differences between SS and Control- (Ct) -derived SGEC. Proteome analysis of cultured SGEC from five SS patients and four Ct was performed in a label-free quantitation format (LFQ). Electron microscopy was applied for analysis of the mitochondrial ultrastructure of SGEC in minor salivary gland sections from six SS patients and four Ct. Four hundred seventy-four proteins were identified differentially abundant in SS- compared to Ct-SGEC. After proteomic analysis, two distinct protein expression patterns were revealed. Gene ontology (GO) pathway analysis of each protein block revealed that the cluster with highly abundant proteins in SS-SGEC showed enrichment in pathways associated with membrane trafficking, exosome-mediated transport and exocytosis as well as innate immunity related mainly to neutrophil degranulation. In contrast, the low abundance protein cluster in SS-SGEC was enriched for proteins regulating the translational process of proteins related to metabolic pathways associated to mitochondria. Electron microscopy showed decreased total number of mitochondria in SS-SGEC, which appeared elongated and swollen with less and abnormal cristae compared to Ct-SGEC mitochondria. This study defines, for the first time, the core proteomic differences of SGEC between SS and Ct, substantiates the metamorphosis of SGEC into an innate immune cell and reveals that these cells are translationally shifted towards metabolism rewiring. These metabolic alterations are related mainly to mitochondria and are mirrored in situ with heavy morphological changes.

Acute effect of smoking and smoking abstinence on energy intake and appetite-related hormones blood concentrations.

The effect of smoking on energy balance and the underlying mechanisms are not fully understood. The aim of the present study is to examine the acute effect of smoking and its abstinence on energy intake, subjective feelings of appetite and related hormones. Fourteen healthy smokers participated in a randomized, crossover study consisting of two trials: the Cigarette trial (participants smoked two cigarettes of their brand within 15min) and the Sham trial (they were asked to hold the cigarette as smoking, but without lighting it). After 45min the participants were offered an ad libitum variety of snacks, and their intake was recorded. Blood samples were taken at fasting, before the ad libitum meal and 1h after and were analyzed for obestatin, ghrelin, glucagon-like peptide-1, cholecystokinin and insulin levels. Subjective feelings of hunger, satiety and desired to eat, as well as smoking craving were evaluated by visual analog scales. Mean energy intake at the ad libitum meal was 825±310kcal in the Sham trial and 673±245kcal in the Cigarette trial (p=0.010). No significant intervention effects were observed for the reported appetite feelings or the appetite-related hormones levels. In conclusion, smoking was found to have an acute effect on dietary intake; this was not explained by changes in the hormonal levels that were evaluated. More research is needed to confirm these results in more prolonged periods of abstinence and explore other pathways through which smoking and its abstinence affect energy balance.

Endoplasmic reticulum stress causes autophagy and apoptosis leading to cellular redistribution of the autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/SSB in salivary gland epithelial cells.

The aim of this study was to examine the levels of endoplasmic reticulum (ER) stress in minor salivary glands, to investigate the interplay between ER stress-induced autophagy and apoptosis in human salivary gland (HSG) cells and to test the effect of ER stress-induced apoptosis on the cellular redistribution of the two major Sjögren's syndrome (SS) autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/Sjögren's syndrome-related antigen B (SSB). Minor salivary gland biopsies from SS patients and sicca controls were examined by immunohistochemistry for the expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/BiP) as an indicator of unfolded protein response (UPR). HSG cells were treated with thapsigargin (TG) and cell viability, autophagy and apoptosis were assessed. Immunoblot was applied to detect the conversion of LC3I to LC3II and the protein levels of GRP78/BiP and X-box binding protein-1 (XBP-1). Apoptosis was evaluated by a single-stranded DNA enzyme-linked immunosorbent assay (ELISA). Ro/SSA and La/SSB localization was visualized using immunofluorescence. GRP78/BiP was expressed by acinar and ductal epithelial cells in salivary glands of patients and sicca controls. TG treatment induced autophagy, as indicated by enhanced protein expression of LC3II. The protein levels of UPR marker XBP-1 were increased after TG treatment, while GRP78/BiP levels were decreased. TG treatment resulted in induction of HSG apoptosis. Ro/SSA and La/SSB autoantigens were localized predominantly to the cytoplasm in resting cells, while they were redistributed to cell membrane and blebs in the apoptotic cells. In conclusion, ER stress is activated in minor salivary gland epithelial cells from SS patients and controls. ER stress-induced apoptosis in HSG cells leads to cell surface and apoptotic blebs relocalization of Ro/SSA and La/SSB autoantigens.

Association between circulating levels of adiponectin and indices of bone mass and bone metabolism in middle-aged post-menopausal women.

BACKGROUND: Adiponectin, a fat derived cytokine, is a potential independent contributor to bone mineral density (BMD); however, its action on bone metabolism in humans is still unclear. AIM: The aim of this study was to investigate the relationship of adiponectin with bone mass indices and bone metabolic markers in middle-aged post-menopausal women without diabetes. SUBJECTS AND METHODS: A random sample consisted of 81 post-menopausal women (age range 45-61 yr, osteopenic/osteoporotic no.=43) was studied. Lumbar-spine BMD (BMD(L2-L4)) and total-body bone mineral content (TBBMC) were measured with dual X-ray absorptiometry. Plasma levels of total and high-molecular weight (HMW) adiponectin, osteoprotegerin (OPG), soluble receptor activator of nuclear factor-κB ligand (sRANKL) and IGF-I were determined. RESULTS: No association was observed between total or HMW adiponectin and BMD(L2-L4) or TBBMC. On the contrary, adiponectin levels were positively associated with OPG levels (partial r=0.276, p=0.015) and negatively with IGF-I (partial r=-0.438, p<0.001), in multiple regression models after adjustment for potential confounders. HMW adiponectin showed a negative association with IGF-I (partial r=-0.266, p=0.049) in the multiple regression models but not with OPG, TBBMC or BMD(L2-L4). CONCLUSIONS: Although we failed to show statistically significant association between circulating adiponectin levels and indices of bone mass in women during the postmenopausal period, we showed significant associations with OPG and IGF-I levels, suggesting an anabolic role of adiponectin, which may contribute in the understanding of the interplay between adipose tissue-derived hormones and bone metabolism.

Osteoprotegerin and nuclear factor-kappaB ligand are associated with leptin and adiponectin levels, in apparently healthy women.

OBJECTIVES: We investigated the relationship between the major bone turnover markers, i.e., Osteoprotegerin (OPG) and Receptor activator of nuclear factor-kappaB ligand (RANKL) and serum adipokines (leptin, adiponectin) levels in a sample of apparently healthy women. METHODS: A random sample which consisted of 80 females (18-71 years) was studied. Elisa method was used to measure the OPG, RANKL and the leptin, adiponectin levels in females' serum. RESULTS: OPG values were inversely correlated with leptin (rho = -0.38, p = 0.002) and positively correlated with age (rho = 0.27, p=0.01) and body mass index (rho = 0.29, p=0.009). RANKL values were inversely correlated with adiponectin (rho = -0.23, p = 0.06) and age (rho = -0.30, p=0.01). Additionally, OPG was higher in post- as compared to pre-menopausal women. Further data analysis adjusting for potential confounders revealed that the OPG/RANKL ratio was positively associated with adiponectin and inversely associated with leptin levels independent of the effect of age, body mass index and menopausal status. CONCLUSIONS: These results reveal that leptin circulating levels are inversely associated with serum OPG/RANKL ratio among healthy women.

Mechanisms of the action of zoledronic acid on human MG-63 osteosarcoma cells.

The aim of our study was to analyze the action of zoledronic acid on MG-63 human osteosarcoma cells. The proliferation of MG-63 cells was inhibited by either continuous or pulsatile exposures of zoledronic acid in a dose-dependent manner (10-250 microM). Zoledronic acid did not produce evidence of MG-63 cell death when administered at 100 mM for 48 hours, but only after exposure of 96 hours. Zoledronic acid (100 microM) increased the distribution of MG-63 cells in G0/G1 phase, however, it did not increase the adriamycin-induced apoptosis. In addition, zoledronic acid action was partially neutralized by exogenous administration of geranylgeranyl pyrophosphate (GGPP), but not by farnesyl pyrophosphate (FPP). Furthermore, zoledronic acid resulted in the attenuation of the prenylated form of Ras. Zoledronic acid and EDTA increased fluorescence of Fluo-3 loaded MG-63 cells in a similar pattern. This increase was owing to the release of Ca2+ from intracellular stores since zoledronic acid failed to reveal such a change to intracellular Ca2+ when cells were previously treated with 1 mM caffeine. Moreover, zoledronic acid significantly decreased the expression of estrogen receptor alpha (ERalpha) whereas it did not change significantly the expression of estrogen receptor beta (ERbeta) in MG-63 cells. These data suggest that zoledronic acid can control the proliferation and the differentiation of osteosarcoma-like cells.

Microarray analysis of survival pathways in human PC-3 prostate cancer cells.

BACKGROUND: Insulin-like growth factor 1 (IGF-1), transforming growth factor beta 1 (TGFbeta1), and interleukin 6 (IL-6), act as survival factors inhibiting chemotherapy-induced apoptosis in PC-3 human prostate cancer cells, in vitro. MATERIALS AND METHODS: To study the intracellular pathways activated by these survival factors we performed a comparative genomic analysis using oligonucleotide microarray chips. A validation by real time-PCR was also performed for the genes of interest. RESULTS: The expression data derived were analysed using various normalization algorithms. The differentially expressed genes were clustered and their ontological annotations were statistically tested to provide evidence for possible deregulated biological processes on the action of the aforementioned survival factors. Emphasis was given on the regulation and the role of the genes AKR1C1, SDPR and GADD45B in the survival pathways of prostate cancer cells, whose expression was also validated by real time-PCR. CONCLUSION: The overall analyses reveal an overrepresentation of differentially expressed genes related to cellular processes such as cell cycle regulation, lipid metabolism and steroid biosynthesis.

Bone microenvironment-related growth factors, zoledronic acid and dexamethasone differentially modulate PTHrP expression in PC-3 prostate cancer cells.

Bone metastasis microenvironment-related growth factors such as insulin-like growth factor 1 (IGF-1), transforming growth factor beta 1 (TGF-beta1), basic fibroblast growth factor (bFGF) and interleukin 6 (IL-6) show survival factor activity, thereby inhibiting chemotherapy-induced apoptosis of PC-3 prostate cancer cells in vitro. Recently, zoledronic acid has been shown to induce apoptosis in PC-3 prostate cancer cells while overexpression of parathyroid hormone-related protein (PTHrP) inhibits serum deprivation-induced apoptosis in PC-3 cells. Consequently, we have investigated whether IGF-1, TGF-beta1, bFGF, IL-6, zoledronic acid and/or dexamethasone affect the expression of the PTHrP and type I PTH/PTHrP receptor (PTH.1R) in PC-3 prostate cancer cells using relative quantitative PCR and real-time PCR (expression at mRNA level) and immunocytochemical and immunofluorescence analysis (expression at protein level). Our data show that IGF-1, TGF-beta1, bFGF and IL-6 increase PTHrP mRNA expression and its perinuclear localization, while zoledronic acid (50 muM, 100 muM for 24 h and 48 h) and dexamethasone suppress PTHrP expression in PC-3 cells. We did not detect any appreciable change of the PTH.1R expression due to IGF-1, TGF- beta1, bFGF, IL-6, zoledronic acid or dexamethasone in PC-3 cells. Therefore, it is conceivable that bone metastasis microenvironment-related survival factor/anti-apoptotic activity and zoledronic acid anticancer action/pro-apoptotic activity on PC-3 cells is mediated, at least in part, by differential modulation of PTHrP expression.

Prostate cancer cell survival pathways activated by bone metastasis microenvironment.

The development of resistance to anti-cancer therapies in bones is a major hurdle preventing long-lasting clinical responses to anti-cancer therapies in hormone refractory prostate cancer. Herein, we present the major signal transduction pathways, which are activated in prostate cancer cells residing at bone metastasis microenvironment. These intracellular signal transduction pathways can inhibit anti-cancer therapy-induced apoptosis of metastatic prostate cancer cells, thereby optimizing their survival, locally. Employment of this knowledge in a clinical setting provides the conceptual framework for the development of bone-targeted therapies for advanced prostate cancer. Indeed, bone metastasis microenvironment-targeted therapies illustrate a novel paradigm in cancer treatment: anti-tumor treatment strategies may not only aim at directly inducing cancer cell apoptosis, but can also target the tumor metastasis microenvironment, and neutralize the protection it confers on metastatic cancer cells.

Endocrine/paracrine/autocrine survival factor activity of bone microenvironment participates in the development of androgen ablation and chemotherapy refractoriness of prostate cancer metastasis in skeleton.

Bone is the most frequent site of metastases of prostate cancer and is almost always the first and frequently the only site of metastases where disease will progress to stage D3. In addition, the number of skeletal metastatic foci is the most powerful independent prognostic factor of limited response to hormone ablation therapy and poor survival of patients with advanced prostate cancer. Furthermore, disease progression frequently occurs in the osteoblastic metastases, even though androgen ablation therapy still provides adequate and sustained control of disease at the primary site. Notably, the management of metastatic disease onto bones has traditionally relied on therapeutic modalities, which almost exclusively aim at directly inducing cancer cell death. However, accumulating pieces of evidence, from both the clinical and the basic research front, point to major limitations of this conventional approach. The in vivo response of malignant cells to anticancer therapies is directly influenced by the local microenvironment in which they metastasize. In particular, organ sites frequently involved in metastatic diseases, such as the bones, appear to confer to metastatic cells protection from anticancer drug-induced apoptosis. This protection is mediated by soluble growth factors and cytokines released by the normal cellular constituents of the host tissue microenvironment. The characterization of bone microenvironment-related survival factors has led to the development of a novel hormone manipulation which can re-introduce clinical responses in patients with stage D3 prostate cancer.