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Therapy-induced senescence (TIS) is a major cellular response to anticancer therapies. While induction of a persistent growth arrest would be a desirable outcome in cancer therapy, it has been shown that, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, likely contributing to tumor relapse. Notably, cells that escape from TIS acquire a plastic, stem cell-like phenotype. The metabolic dependencies of cells that evade senescence have not been thoroughly studied. In this study, we show that glutamine depletion inhibits escape from TIS in all cell lines studied, and reduces the stem cell subpopulation. In line with a metabolic reliance on glutamine, escaped clones overexpress the glutamine transporter SLC1A5. We also demonstrate a central role of glutamine synthetase that mediates resistance to glutamine deprivation, conferring independence from exogenous glutamine. Finally, rescue experiments demonstrate that glutamine provides nitrogen for nucleotides biosynthesis in cells that escape from TIS, but also suggest a critical involvement of glutamine in other metabolic and non-metabolic pathways. On the whole, these results reveal a metabolic vulnerability of cancer stem cells that recover proliferation after exposure to anticancer therapies, which could be exploited to prevent tumor recurrence.

Prostate Cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in males and the fifth leading cause of death worldwide. The majority of PCas are androgen-sensitive, with a significant up-regulation of Androgen Receptor (AR) that causes a stimulatory effect on growth and progression of cancer cells. For this reason, the first-line therapy for PCa is androgen ablation, even if it ultimately fails due to the onset of hormone-refractory state, in which the malignant cells do not sense the androgen signal anymore. Besides androgens, a growing number of evidence suggests that Thyroid Hormones (THs) mediate tumor-promoting effects in a variety of human cancers, as Epithelial-to-Mesenchymal Transition (EMT), invasion and metastasis and also stimulation of angiogenesis and tumor metabolism. Moreover, epidemiological studies demonstrated an increased risk for PCa in patients with lower levels of Thyreotropin (TSH). Here, we investigated if intracellular TH metabolism affects Benign Prostatic Hyperplasia (BPH) and PCa formation and progression. We found that the intracellular TH metabolism is a crucial determinant of PCa behavior. We observed that a dynamic stage-specific expression of the THs modulating enzymes, the deiodinases, is required for the progression of BPH to PCa malignancy. By acting simultaneously on epithelial cancer cells and fibroblasts, THs exert a proliferative and pro-inflammatory effect cooperating with androgens. These findings suggest that androgens and THs may interplay and mediate a coordinate effect on human PCa formation and progression. In light of our results, future perspective could be to explore the potential benefits of THs intracellular modulators aimed to counteract PCa progression.

Therapy-induced senescence (TIS or therapy-induced premature senescence) is a key cellular program triggered in the course of cancer radiotherapy and chemotherapy with genotoxic drugs, both in cancer cells and in normal cells, whose activation critically affects the outcome of cancer therapy. Drug-induced senescent cells undergo a permanent cell cycle arrest, acquire distinctive morphological and biochemical alterations, and an enhanced secretory ability, referred to as senescence-associated secretory phenotype (SASP). The transcription factor NF-κB acts as a master regulator of the SASP, driving the expression of senescence-associated secretome components.Here we describe protocols for the establishment of a tetracycline-regulated cell system for the investigation of the role of NF-κB in TIS. We also describe protocols routinely used in our laboratory, to investigate TIS in this Tet-On inducible expression system. Finally, we describe techniques for the validation of TIS induction.

Therapy-induced senescence (TIS or therapy-induced premature senescence) is a key cellular program triggered in the course of cancer radiotherapy and chemotherapy with genotoxic drugs, both in cancer cells and in normal cells, whose activation critically affects the outcome of cancer therapy. Drug-induced senescent cells undergo a permanent cell cycle arrest, acquire distinctive morphological and biochemical alterations, and an enhanced secretory ability, referred to as senescence-associated secretory phenotype (SASP). The transcription factor NF-κB acts as a master regulator of the SASP, driving the expression of senescence-associated secretome components. Here we describe protocols for the establishment of a tetracycline-regulated cell system for the investigation of the role of NF-κB in TIS. We also describe protocols routinely used in our laboratory, to investigate TIS in this Tet-On inducible expression system. Finally, we describe techniques for the validation of TIS induction.

Cellular senescence is a stress response characterized by a permanent loss of proliferative ability. Different types of senescence reflect different forms of stress. Although cellular senescence plays a key role in maintaining tissue homeostasis and in suppressing tumorigenesis, it is widely appreciated that accumulation of senescent cells in tissue contributes to development of age-related dysfunctions and limits the efficacy of chemo- and radiotherapy. Consequently, novel therapeutic strategies targeting senescent cells, or their pro-inflammatory secretome, have been recently developed. The application of these novel therapeutic approaches requires biomarkers for in vivo detection of senescent cells for ageing and age-associated diseases, as well as for therapy-induced senescence in cancer patients. Aptamers are short oligonucleotide or peptide sequences able to bind with high affinity and specificity a wide range of cellular targets, including cell surface epitopes, thanks to their unique three-dimensional folding. Oligonucleotide aptamers have been widely applied for the discovery of biomarkers, since the early 1990s, and Affimers have recently emerged as valuable tools for biomarker discovery. In this chapter we briefly introduce applications of aptamer technologies for recognition of cell surface antigens and suggest that aptamers provide the perfect technology to identify novel senescence-specific biomarkers.

Intake of large amounts of added sweeteners has been associated with the pathogenesis of cardiometabolic risk. Several studies have shown that fructose increases the cardiovascular risk by modulating endothelial dysfunction and promoting atherosclerosis. Recently, a potential role for fructose in cardiovascular thrombosis has been suggested but with controversial results. Tissue factor (TF) plays a pivotal role in the pathophysiology of cardiovascular thrombosis by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of fructose, in a concentration range usually observed in the plasma of patients with increased cardiovascular risk, on TF in human umbilical endothelial cells (HUVECs). Cells were stimulated with increasing concentrations of fructose (0.25, 1 and 2.5 mM) and then processed to evaluate TF-mRNA levels by real-time PCR as well as TF expression/activity by FACS analysis and procoagulant activity. Finally, a potential molecular pathway involved in modulating this phenomenon was investigated. We demonstrate that fructose induces transcription of mRNA for TF. In addition, we show that this monosaccharide promotes surface expression of TF that is functionally active. Fructose effects on TF appear modulated by the oxygen free radicals through activation of the transcription factor NF-κB since superoxide dismutase and NF-κB inhibitors suppressed TF expression. Data of the present study, although in vitro, indicate that fructose, besides promoting atherosclerosis, induces a prothrombotic phenotype in HUVECs, thus indicating one the mechanism(s) by which this sweetener might increase cardiometabolic risk.

IL-17 is a proinflammatory cytokine that promotes the expression of different cytokines and chemokines via the induction of gene transcription and the posttranscriptional stabilization of mRNAs. In this study, we show that IL-17 increases the half-life of the Zc3h12a mRNA via interaction of the adaptor protein CIKS with the DEAD box protein DDX3X. IL-17 stimulation promotes the formation of a complex between CIKS and DDX3X, and this interaction requires the helicase domain of DDX3X but not its ATPase activity. DDX3X knockdown decreases the IL-17–induced stability of Zc3h12a without affecting the stability of other mRNAs. IKKε, TNFR-associated factor 2, and TNFR-associated factor 5 were also required to mediate the IL-17–induced Zc3h12a stabilization. DDX3X directly binds the Zc3h12a mRNA after IL-17 stimulation. Collectively, our findings define a novel, IL-17–dependent mechanism regulating the stabilization of a selected mRNA.

Premature or drug-induced senescence is a major cellular response to chemotherapy in solid tumors. The senescent phenotype develops slowly and is associated with chronic DNA damage response. We found that expression of wild-type p53-induced phosphatase 1 (Wip1) is markedly down-regulated during persistent DNA damage and after drug release during the acquisition of the senescent phenotype in carcinoma cells. We demonstrate that down-regulation of Wip1 is required for maintenance of permanent G2 arrest. In fact, we show that forced expression of Wip1 in premature senescent tumor cells induces inappropriate re-initiation of mitosis, uncontrolled polyploid progression, and cell death by mitotic failure. Most of the effects of Wip1 may be attributed to its ability to dephosphorylate p53 at Ser15 and to inhibit DNA damage response. However, we also uncover a regulatory pathway whereby suppression of p53 Ser15 phosphorylation is associated with enhanced phosphorylation at Ser46, increased p53 protein levels, and induction of Noxa expression. On the whole, our data indicate that down-regulation of Wip1 expression during premature senescence plays a pivotal role in regulating several p53-dependent aspects of the senescent phenotype. Background: Wip1 is a phosphatase involved in DNA-damage response. Results: Wip1 expression is down-regulated in premature senescent cancer cells. Failure to down-regulate Wip1 expression results in cell death and polyploidy. Conclusion: Wip1 down-regulation is important for maintenance of permanent cell cycle arrest in premature senescent tumor cells. Significance: These findings improve our understanding of the mechanism by which Wip1 promotes tumor progression.

Context: We have previously identified neutrophil gelatinase-associated lipocalin (NGAL) as one of the genes mediating the oncogenic activity of nuclear factor-?B in human anaplastic thyroid carcinomas (ATCs). Objectives: To further investigate the role of NGAL in thyroid cancer, we established NGAL knocked-down and NGAL overexpressing ATC cell lines. Results: We found that the ability of NGAL knocked-down cells to degrade Matrigel in a transwell invasion assay and to form lung metastasis in nude mice was decreased. Because NGAL binds matrix metalloproteinase-9 (MMP-9), to form a macromolecular complex involved in the regulation of metastatic spread of cancer cells and given the strong expression of both genes in tissue specimens from human ATCs, we analyzed the MMP-9 enzymatic activity in NGAL-null ATC cells. Enzymatic immunoassays show that MMP-9 activity is reduced in NGAL-null ATC cells, even if its expression is not affected by NGAL inhibition. Ectopic expression of NGAL in an ATC cell line not expressing NGAL determines an increase of its metastatic property. The use of a mutated form of NGAL, unable to bind MMP-9, has no positive effect on the invasive potential of ATC cells and does not improve the MMP-9 enzymatic activity. Conclusions: Our results indicate NGAL as a novel target of nuclear factor-?B prometastatic activity in thyroid cancer through enhancement of MMP-9 enzymatic activity.

Introduction Adipocytes are nowadays recognized as cells able to produce and secrete a large variety of active substances with direct effects on vascular cells, known as adipokines. Visfatin is a recently identified adipokine not yet completely characterized for its pathophysiological role in cardiovascular disease. Increased levels of visfatin are measurable in the plasma of patients with coronary artery disease and specifically in those with acute coronary syndromes (ACS). Several studies have indicated that Tissue Factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of visfatin on TF in human coronary endothelial cells (HCAECs). Methods HCAECs were stimulated with visfatin in a concentration range usually measurable in plasma of patients with ACS and than processed to evaluate TF-mRNA levels as well as TF expression/activity. Finally, the role of NF-κB pathway was investigated. Results We demonstrate that visfatin induces transcription of mRNA for TF by Real Time PCR. In addition, we show that this adipokine promotes surface expression of TF that is functionally active since we measured increased procoagulant activity. Visfatin effects on TF appear modulated by the activation of the transcription factor, NF-κB, since NF-κB inhibitors suppressed TF expression. Finally, we show that the nicotinamide phopsphoribosyltransferase enzymatic activity of visfatin seems to play a pivotal role in modulating the NF-κB driven regulation of TF. Discussion Data of the present study, although in vitro, indicate that visfatin, at doses measurable in ACS patient plasma, induces a procoagulant phenotype in human coronary endothelial cells by promoting TF expression. These observations support the hypothesis that this adipokine might play a relevant role as an active partaker in athero-thrombotic disease.

Context: We have recently shown that nuclear factor (NF)-κB activity is constitutively elevated in anaplastic human thyroid carcinomas. The inhibition of NF-κB in the anaplastic thyroid carcinoma cell line (FRO) leads to increased susceptibility to apoptosis induced by chemotherapeutic drugs and to the block of oncogenic activity. Objectives: To understand better the molecular mechanisms played by NF-κB in thyroid oncogenesis, we performed a differential proteomic analysis between FRO transfected with a superrepressor form of inhibitor of κBα (IκBαM) and the parental counterpart (FRO Neo cells). Results: Differential proteomic analysis revealed that the retinoblastoma-associated protein 48 (RbAp48) is down-regulated in the absence of functional NF-κB. Immunohistochemical analysis of normal and pathological human thyroid specimens confirmed that RbAp48 is strongly overexpressed in primary human carcinomas. Reduction of RbAp48 expression using small interfering RNA determined the suppression of tumorigenicity, very likely due to the decrease of their growth rate rather than to an increased susceptibility to apoptosis. In addition, we showed that NF-κB, at least in part, transcriptionally controls RbAp 48. A functional NF-κB consensus sequence was located within the promoter region of RbAp48 human gene, and embryonic fibroblasts isolated from the p65 knockout mouse (murine embryonic fibroblasts p65−/−) showed decreased expression of RbAp48. Conclusion: Our results show that RbAp48 is a NF-κB-regulated gene playing an important role in thyroid cancer cell autonomous proliferation.

OBJECTIVE: We recently reported that the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2b is the SERCA form preferentially expressed in rat thyroid. Moreover, SERCA2b expression dramatically decreases in virally transformed, highly tumorigenic, PC Cl3 thyroid cells. These results suggest that, in the thyroid, SERCA2b, in addition to its housekeeping role, is linked to differentiation and is a regulated gene. We therefore sought to study the effect of TSH, the main regulator of thyroid function, on SERCA2b expression and activity. METHODS: PC Cl3 cells were hormone starved in low-serum medium and stimulated for long (48 h) or short (1, 2 and 4 h) times. SERCA2b expression and activity were evaluated by Northern and Western blots, Ca2+-ATPase activity and Ca2+ store content. RESULTS: In PC Cl3 cells, SERCA2b mRNA and protein were induced twofold by a 48-h long treatment with TSH. Long-term elevation (48 h) of intracellular cAMP levels, by forskolin or 8-Br-cAMP, had similar effects on SERCA2b mRNA and protein. We also measured Ca2+-ATPase activity and Ca2+ store content. Both long (48 h) and short (0.5-1 h) treatments with TSH, forskolin or 8-Br-cAMP induced a marked increase of SERCA2b activity. This effect was completely abolished by H89, a specific inhibitor of cAMP-dependent protein kinase A (PKA). TSH and 8-Br-cAMP increased Ca2+ store content after both long (48 h) and short (1-2 h) treatments. CONCLUSIONS: These data suggested that TSH/cAMP acts as an important regulator of both SERCA2b expression and activity in the thyroid system, through PKA activation.

Perturbing the endoplasmic reticulum homeostasis of thyroid cell lines with thapsigargin, a specific inhibitor of the sarcoendoplasmic reticulum Ca(2+) adenosine triphosphatases, and tunicamycin, an inhibitor of the N-linked glycosylation, blocked Tg in the endoplasmic reticulum. This event was signaled outside the endoplasmic reticulum and resulted in activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase and nuclear factor-kappa B (NF-kappa B) stress response pathways. Activation of the JNK/stress-activated protein kinase signaling pathway was assessed by measuring the amount of phospho-JNK and the activity of JNK by kinase assays. Activation of the NF-kappa B signaling pathway was assessed by measuring the level of inhibitory subunit I kappa B alpha, DNA binding, and transcriptional activity of NF-kappa B. Cycloheximide treatment, at a dose able to profoundly inhibit protein synthesis in FRTL-5 cells, obliterated the decrease in the level of the inhibitory subunit I kappa B alpha produced by thapsigargin and tunicamycin. Therefore, protein synthesis was required to generate a signal from stressed endoplasmic reticulum. This substantiates the hypothesis that endoplasmic reticulum retention of newly synthesized Tg and other cargo (secretory and membrane) proteins functions upstream of signal activation. Dominant negative TNF receptor-associated factor 2 (TRAF2) inhibited activation of NF-kappa B, which was also inhibited in embryonic fibroblasts derived from TRAF2(-/-) mice, respect to their normal counterpart. These data extend the recent demonstration that TRAF2 mediated JNK activation in response to endoplasmic reticulum stress and strongly strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals.

Follicular thyroglobulin (TG) decreases expression of the thyroid-restricted transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, thereby suppressing expression of the sodium iodide symporter, thyroid peroxidase, TG, and thyrotropin receptor genes (Suzuki, K., Lavaroni, S., Mori, A., Ohta, M., Saito, J., Pietrarelli, M., Singer, D. S., Kimura, S., Katoh, R., Kawaoi, A. , and Kohn, L. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 95, 8251-8256). The ability of highly purified 27, 19, or 12 S follicular TG to suppress thyroid-restricted gene expression correlates with their ability to bind to FRTL-5 thyrocytes and is inhibited by a specific antibody to the thyroid apical membrane asialoglycoprotein receptor (ASGPR), which is related to the ASGPR of liver cells. Phosphorylating serine/threonine residues of TG, by autophosphorylation or protein kinase A, eliminates TG suppression and enhances transcript levels of the thyroid-restricted genes 2-fold in the absence of a change in TG binding to the ASGPR. Follicular TG suppression of thyroid-restricted genes is thus mediated by the ASPGR on the thyrocyte apical membrane and regulated by a signal system wherein phosphorylation of serine/threonine residues on the bound ligand is an important component. These data provide a hitherto unsuspected role for the ASGPR in transcriptional signaling, aside from its role in endocytosis. They establish a functional role for phosphorylated serine/threonine residues on the TG molecule.

Adipocytes are nowadays recognized as cells able to produce and secrete a large variety of active substances with direct effects on vascular cells, known as adipokines. Visfatin is a recently identified adipokine not yet completely characterized for its pathophysiological role in cardiovascular disease. Increased levels of visfatin are measurable in the plasma of patients with coronary artery disease and specifically in those with acute coronary syndromes (ACS). Several studies have indicated that Tissue Factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of visfatin on TF in human coronary endothelial cells (HCAECs).HCAECs were stimulated with visfatin in a concentration range usually measurable in plasma of patients with ACS and than processed to evaluate TF-mRNA levels as well as TF expression/activity. Finally, the role of NF-κB pathway was investigated.We demonstrate that visfatin induces transcription of mRNA for TF by Real Time PCR. In addition, we show that this adipokine promotes surface expression of TF that is functionally active since we measured increased procoagulant activity. Visfatin effects on TF appear modulated by the activation of the transcription factor, NF-κB, since NF-κB inhibitors suppressed TF expression. Finally, we show that the nicotinamide phopsphoribosyltransferase enzymatic activity of visfatin seems to play a pivotal role in modulating the NF-κB driven regulation of TF.Data of the present study, although in vitro, indicate that visfatin, at doses measurable in ACS patient plasma, induces a procoagulant phenotype in human coronary endothelial cells by promoting TF expression. These observations support the hypothesis that this adipokine might play a relevant role as an active partaker in athero-thrombotic disease.

Cancer is a multistep process during which cells acquire genetic alterations that drive the progressive transformation of normal cells into highly malignant cells. Self-sufficiency in growth, insensitivity to anti-growth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis, are signatures of transformed cells. NF-kappaB is a key actor in tumorigenesis given its ability to control the expression and the function of a number of genes involved in these processes. Indeed, constitutive activation of NF-kappaB is a common feature of many human tumors, while its sustained activation during inflammation predisposes normal cells to neoplastic transformation. Since suppression of NF-kappaB has been shown to inhibit oncogenic potential of transformed cells, targeting it should be effective in the prevention and treatment of cancer.

The rat hepatic lectin (RHL)-1 is the major component of the rat liver asialoglycoprotein receptor (ASGPr), a membrane receptor highly expressed on the basolateral side of hepatocytes, which mediates endocytosis of serum desialated glycoproteins. We have recently shown that RHL-1 is expressed in rat thyroid tissue and thyroid differentiated cell lines. Both in vitro and in vivo assays show that thyrotropin up-regulates thyroid RHL-1 expression, while neoplastic transformation of thyroid cells exerts a down-regulation of receptor expression. Moreover, RHL-1 expressed on the surface of differentiated thyroid cells is able to bind thyroglobulin (Tg), the macromolecular site of synthesis and storage of thyroid hormones. In the present work, we demonstrate, by immunohistochemistry analysis, that RHL-1 is localized on the apical surface of thyrocytes, at a variance with its basolateral localization on hepatocytes. Moreover, albeit its expression in thyroid is less abundant than in liver, the receptor is able to bind asialorosomucoid (ASOR), the best-known ligand of hepatic ASGPr, and to mediate endocytosis of a significative amount of Tg on the surface of differentiated PC Cl3 thyroid cells. Taken together, the data suggest that RHL-1, even if expressed in thyroid at lower levels than in liver, could serve as a receptor for endocytosis of colloidal Tg and, likely, for its delivery to lysosomes.

Nuclear factor kappaB (NF-kappaB) plays a pivotal role in numerous cellular processes, including stress response, inflammation, and protection from apoptosis. Therefore, the activity of NF-kappaB needs to be tightly regulated. We have previously identified a novel gene, named CIKS (connection to IkappaB-kinase and SAPK), able to bind the regulatory sub-unit NEMO/IKKgamma and to activate NF-kappaB. Here, we demonstrate that CIKS forms homo-oligomers, interacts with NEMO/IKKgamma, and is recruited to the IKK-complex upon cell stimulation. In addition, we identified the regions of CIKS responsible for these functions. We found that the ability of CIKS to oligomerize, and to be recruited to the IKK-complex is not sufficient to activate the NF-kappaB In fact, a deletion mutant of CIKS able to oligomerize, to interact with NEMO/IKKgamma, and to be recruited to the IKK-complex does not activate NF-kappaB, suggesting that CIKS needs a second level of regulation to efficiently activate NF-kappaB. (C) 2003 Elsevier Inc. All rights reserved.

We have recently identified a novel gene, named CIKS ( C onnection to IK K-complex and S APK), able to activate the transcription factor NF-κB, after interaction with the regulatory subunit NEMO/IKKγ of IKK complex, and the stress-activated protein kinase (SAPK)/JNK. CIKS mRNA is ubiquitously expressed, although its levels differ greatly among different tissues. The aim of this study is to identify and characterize the promoter region of CIKS gene and to analyse the regulation of its expression by different cytokines. The transcription start site of CIKS mRNA was mapped both by primer extension and by a polymerase chain reaction (PCR)-based strategy. The proximal 5′-flanking region of CIKS gene was ‘TATA-less’, but contained other consensus promoter elements including an initiator (Inr), ‘GC’ and ‘CAAT’ boxes. Transfection of luciferase reporter plasmids containing 1.8 kb of the 5′-flanking region increased luciferase activity in epithelial MDCK cells, but not in endothelial HUVEC cells. Deletion analysis identified a sequence from −464 to −220 bp of the 5′-flanking region of CIKS gene essential for basal promoter activity in MDCK cells. Competitive reverse transcriptase–PCR, Northern and Western blot assays showed that different cytokines, such as tumor necrosis factor (TNF)-α, Interleukin (IL)-1β and transforming growth factor (TGF)-β, dramatically increased CIKS mRNA expression in HeLa cells. We conclude that the proximal 5′-flanking region of CIKS gene contains a functional promoter and binding sites for nuclear proteins leading to its basal transcription. Moreover, we demonstrate that the expression of CIKS is up-regulated by different cytokines.

During its initial folding in the endoplasmic reticulum (ER), newly synthesized thyroglobulin (Tg) is known to interact with calnexin and other ER molecular chaperones, but its interaction with calreticulin has not been examined previously. In the present study, we have investigated the interactions of endogenous Tg with calreticulin and with several other ER chaperones. We find that, in FRTL-5 and PC-Cl3 cells, calnexin and calreticulin interact with newly synthesized Tg in a carbohydrate-dependent manner, with largely overlapping kinetics that are concomitant with the maturation of Tg intrachain disulphide bonds, preceding Tg dimerization and exit from the ER. Calreticulin co-precipitates more newly synthesized Tg than does calnexin; however, using two different experimental approaches, calnexin and calreticulin were found in ternary complexes with Tg, making this the first endogenous protein reported in ternary complexes with calnexin and calreticulin in the ER of live cells. Depletion of Ca2+ from the ER elicited by thapsigargin (a specific inhibitor of ER Ca2+-ATPases) results in retention of Tg in this organelle. Interestingly, thapsigargin treatment induces the premature exit of Tg from the calnexin/calreticulin cycle, while stabilizing and prolonging interactions of Tg with BiP (immunoglobulin heavy chain binding protein) and GRP94 (glucose-regulated protein 94), two chaperones whose binding is not carbohydrate-dependent. Our results suggest that calnexin and calreticulin, acting in ternary complexes with a large glycoprotein substrate such as Tg, might be engaged in the folding of distinct domains, and indicate that lumenal Ca2+ strongly influences the folding of exportable glycoproteins, in part by regulating the balance of substrate binding to different molecular chaperone systems within the ER.

Maintaining a high Ca(2+) concentration in the lumen of the endoplasmic reticulum (ER), by the action of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs), is important in many cellular processes, such as Ca(2+)-mediated cytosolic signaling in response to extracellular stimuli, cell growth and proliferation, and synthesis, processing and folding of ER-translated proteins. In the thyroid gland, SERCAs have not been studied yet, and there is little information available on general problems such as the expression of SERCAs following neoplastic transformation. In this study we investigated the expression of SERCA2b and SERCA3 in rat thyroid tIssue and, in addition, in normal and transformed rat thyroid cell lines. RT-PCR and Northern blot assays showed that SERCA2b is the SERCA form preferentially expressed in the thyroid. In rat thyroid, SERCA2b mRNA was expressed at a higher level than that of other non-muscle tIssues such as liver or spleen, but at much lower level than in brain. On the other hand, SERCA3 mRNA was not detected in thyroid by Northern blot analysis, or barely detected by RT-PCR assays. We also studied the SERCA2b expression pattern in PC Cl3 thyroid cells transformed by several oncogenes that induce different degrees of malignancy and dedifferentiation. RT-PCR and Northern blot assays showed that SERCA2b mRNA expression dramatically decreased in highly tumorigenic thyroid cells, while expression of glyceraldehyde-3-phosphate dehydrogenase mRNA, a housekeeping gene used as internal control, exhibited no variations. The dramatic down-regulation of SERCA2b expression in fully transformed thyroid cells was also evident by Western blot analysis. Also, following neoplastic transformation of thyroid cells, the enzymatic activity of SERCA2b was reduced in a measure which correlated with the mRNA and protein levels. Therefore, rat thyrocytes expressed intermediate levels of SERCAs, mostly the SERCA2b isoform. This pattern of expression was basically reproduced in fully differentiated thyroid cells in culture and was sensitive to neoplastic transformation.

We have recently shown that the rat hepatic lectin (RHL)-1 subunit of the asialoglycoprotein receptor (ASGPr) is expressed in the PC C13 differentiated thyroid cell line. To investigate in vivo the expression of RHL-1 and the ability of thyrotropin (TSH) to modulate its expression, reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot assays have been performed on thyroid extracts from rats treated with thyroxine (T4) or propylthiouracil (PTU), each of which modulates TSH levels. It is shown that RHL-1 expression is down-regulated by T4 (which decreases serum TSH) and upregulated by PTU (which increases serum TSH), at both mRNA and protein levels. The sensitivity of RHL-1 to neoplastic transformation of thyroid cells has been investigated. The RHL-1 expression pattern has been studied in PC C13 thyroid cells transformed by several oncogenes that induce different degrees of malignancy and dedifferentiation. RT-PCR and Western blot assays show that RHL-1 expression progressively decreases as PC C13 cells acquire a more transformed phenotype. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, a housekeeping gene used as internal control to normalize RHL-1 mRNA content, exhibits no variations in the different PC C13 cell lines used. In addition, we show that both native and asialo-thyroglobulin (Tg) bind RHL-1 in vitro, and native Tg binds RHL-1 on the surface of PC C13 cells. After thyroid cells transformation, the surface expression of RHL-1 is inhibited in a measure that correlates with the mRNA and protein levels. Therefore, the RHL-1 inhibition at the mRNA, protein and plasma membrane expression follows a gradient that parallels the progressive acquisition of the fully transformed phenotype in the PC C13 system. The results reported in the present article, together with our previous data, suggest that RHL-1 expression could be regulated, at least in part, by the same transcription factors involved in the expression of the other molecules characteristic of the thyroid differentiated state.

We have previously reported that the rat hepatic lectin-1 (RHL-1) subunit of rat asialoglycoprotein receptor (ASGPr), the endocytic receptor found on the basolateral surface of hepatocytes, was expressed in rat thyroid tissue and localized on the apical surface of polarized rat thyroid FRT cells. Here we show that PC Cl3 cells, a differentiated rat thyroid cell line, bound thyroglobulin (Tg) via ASGPr. In fact, both the bacterial recombinant carbohydrate recognition domain of RHL-1 (rCRD(RHL-1)) and the anti-rCRD(RHL-1) antibody markedly inhibited (125)I-Tg binding to the cell surface of PC Cl3 cells. Ligand blot assays with deglycosylated Tg show that the rCRD(RHL-1) was able to interact with Tg even after remotion of sugars. The region of Tg involved in the binding to RHL-1 was investigated by ligand blot assays with biotinylated rCRD(RHL-1) on thermolysin-digested native and desialated rat thyroglobulin. It is shown that the rCRD(RHL-1) specifically recognized a thyroglobulin fragment with an apparent M(r) of 68,000, corresponding to the amino-terminal part of the molecule. To our knowledge, this is the first report that attributes to the amino-terminal portion of Tg molecule, containing its earliest and major hormonogenic site, the function of binding to a cell surface receptor of the thyroid. Moreover, we show that oligosaccharides are not the only molecular signals for binding to RHL-1, but amino acidic determinants could also play a role.

Background: Previous studies indicate that platelets and leucocytes might contribute to the development of neointimal hyperplasia following arterial injury. The present study was aimed at further investigating the role of platelets and leucocytes, alone or in combination, in promoting vascular smooth muscle cell (SMC) proliferation in vitro, focusing on the relative contribution of different soluble growth factors released by these cells, and on the ability to induce proto-oncogene expression. such as c-fos. Methods: SMCs from rabbit aortas, made quiescent by serum deprivation, were stimulated with either activated platelets, leucocytes, or both, separated from SMCs by a membrane insert. SMC proliferation was evaluated by measuring the incorporation of H-3-thymidine. The relative contribution of different platelet-derived mediators to SMC growth was evaluated by adding either ketanserin, a 5-HT2 receptor antagonist, R68070, a TxA(2) receptor antagonist, BN52021, a platelet activating factor (PAF) receptor antagonist, and trapidil, a platelet derived growth factor (PDGF) receptor antagonist. The role of different leucocyte sub-populations (neutrophils and monocytes+lymphocytes) was also determined in additional experiments. Results: SMC proliferation was significantly increased by activated platelets to 360+/-9% of control values (P