New Insights into the Evolution and Gene Structure of the Mitochondrial Carrier Family Unveiled by Analyzing the Frequent and Conserved Intron Positions.

Mitochondrial carriers (MCs) belong to a eukaryotic protein family of transporters that in higher organisms is called the solute carrier family 25 (SLC25). All MCs have characteristic triplicated sequence repeats forming a 3-fold symmetrical structure of a six-transmembrane α-helix bundle with a centrally located substrate-binding site. Biochemical characterization has shown that MCs altogether transport a wide variety of substrates but can be divided into subfamilies, each transporting a few specific substrates. We have investigated the intron positions in the human MC genes and their orthologs of highly diversified organisms. The results demonstrate that several intron positions are present in numerous MC sequences at the same specific points, of which some are 3-fold symmetry related. Many of these frequent intron positions are also conserved in subfamilies or in groups of subfamilies transporting similar substrates. The analyses of the frequent and conserved intron positions in MCs suggest phylogenetic relationships not only between close but also distant homologs as well as a possible involvement of the intron positions in the evolution of the substrate specificity diversification of the MC family members.

Imaging Intron Evolution.

Intron evolution may be readily imaged through the combined use of the "dot plot" function of the NCBI BLAST, aligning two sequences at a time, and the Vertebrate "Multiz" alignment and conservation tool of the UCSC Genome Browser. With the NCBI BLAST, an ideal alignment of two highly conserved sequences generates a diagonal straight line in the plot from the lower left corner to the upper right corner. Gaps in this line correspond to non-conserved sections. In addition, the dot plot of the alignment of a sequence with the same sequence after the removal of the Transposable Elements (TEs) can be observed along the diagonal gaps that correspond to the sites of TE insertion. The UCSC Genome Browser can graph, along the entire sequence of a single gene, the level of overall conservation in vertebrates. This level can be compared with the conservation level of the gene in one or more selected vertebrate species. As an example, we show the graphic analysis of the intron conservation in two genes: the mitochondrial solute carrier 21 (SLC25A21) and the growth hormone receptor (GHR), whose coding sequences are conserved through vertebrates, while their introns show dramatic changes in nucleotide composition and even length. In the SLC25A21, a few short but significant nucleotide sequences are conserved in zebrafish, Xenopus and humans, and the rate of conservation steadily increases from chicken/human to mouse/human alignments. In the GHR, a less conserved gene, the earlier indication of intron conservation is a small signal in chicken/human alignment. The UCSC tool may simultaneously display the conservation level of a gene in different vertebrates, with reference to the level of overall conservation in Vertebrates. It is shown that, at least in SLC25A21, the sites of higher conservation are not always coincident in chicken and zebrafish nor are the sites of higher vertebrate conservation.

5' and 3' splicing signals evolution in vertebrates: Analysis in a conserved gene family.

The mitochondrial solute carrier genes (SLC25) are highly conserved during vertebrate evolution. In most SLC25 genes of zebrafish, chicken, mouse, and human, the introns are located at exactly superimposable positions. In these topographically corresponding introns we studied the composition of the initial and terminal hexanucleotides (5'ss and 3'ss) which are instrumental in splicing signaling, focusing on the evolutionary conservation/mutation dynamics of these genetically related sequences. At each position, the per cent conservation of zebrafish individual nucleotides in chicken, mouse and human is proportional to their percent frequency in zebrafish; furthermore, nucleotide mutations are biased in favor of the more represented nucleotides, thus compensating for those highly represented zebrafish nucleotides which have not been conserved. As a result of these evolutionary dynamics, the general nucleotide composition at each position has remained relatively conserved throughout vertebrates. At 5'ss, following the canonical GT, A and G are largely prevailing at position +3, A at +4 and G at +5 (GT[A/G]AGx). At 3'ss, T and C are largely prevailing at positions -6, -5 and -3, preceding the canonical intron terminal AG ([C/T] [C/T]x[C/T]AG). However, the actual composition of the tetranucleotides at 5' and 3' often does not conform to the above scheme. At 5'ss the more canonical sequence is completely expressed in 63% of cases and partially (2 or 1 matches) in 37 % of cases. At 3'ss the more canonical sequence is completely expressed in 71 % of cases and partially (2 or 1 matches) in 29 % of cases. The nucleotide conservation loss (nucleotide mutation) is higher in the evolution from fish to the last common ancestor of birds and mammals (58 %), then diminishes in the successive evolution steps up to the mammalian common ancestor (10 %), and becomes still lower at the divergence of rodents and primates (5 %).

Conservation of Intronic Sequences in Vertebrate Mitochondrial Solute Carrier Genes (Zebrafish, Chicken, Mouse and Human).

The conservation of intronic sequences was studied in the mitochondrial solute carrier (SLC25A*) genes of Zebrafish, Chicken, Mouse and Human. These genes are homologous and the coding sequences have been well conserved throughout Vertebrates, but the corresponding intronic sequences have been extensively re-edited. However, significant segments of Zebrafish introns are conserved in Chicken, Mouse and Human in carriers SLC25A3, SLC25A21, SLC25A25, SLC25A26, and SLC25A36; Chicken intron segments are conserved in Mouse or Human in three additional carriers, namely SLC25A12, SLC25A13, and SLC25A29. Thus, a quota of the intronic sequences of Euteleostomi has been transferred (through Sarcopterygii) to Birds and (through Sarcopterygii and ancestral Mammals) to Mouse and Human. The degree of conservation of Euteleostomi-derived sequences is low and quite similar in Chicken, Mouse and Human (0.23⁻0.27%). The overall degree of conservation of Sarcopterygii-derived sequences in Mammals is higher, and it is significantly higher in Human than in Mouse (4.4% and 3.2%, respectively). Some of the conserved intronic sequences of SLC25A3, SLC25A21, SLC25A25, and SLC25A29 are exonized in some transcript variants of Zebrafish, Chicken, Mouse, and Human and, with minor nucleotide changes, in other Birds or Mammals.

Conservation/Mutation in the Splice Sites of Mitochondrial Solute Carrier Genes of Vertebrates.

The "canonical" introns begin by the dinucleotide GT and end by the dinucleotide AG. GT, together with a few downstream nucleotides, and AG, with a few of the immediately preceding nucleotides, are thought to be the strongest splicing signals (5'ss and 3'ss, respectively). We examined the composition of the intronic initial and terminal hexanucleotides of the mitochondrial solute carrier genes (SLC25A's) of zebrafish, chicken, mouse, and human. These genes are orthologous and we selected the transcripts in which the arrangement of exons and introns was superimposable in the species considered. Both 5'ss and 3'ss were highly polymorphic, with 104 and 126 different configurations, respectively, in our sample. In the line of evolution from zebrafish to chicken, as well as in that from zebrafish to mammals, the average nucleotide conservation in the four variable nucleotides was about 50 % at 5' and 40 % at 3'. In the divergent evolution of mouse and human, the conservation was about 80 % at 5' and 70 % at 3'. Despite these changes, the splicing signals remain strong enough to operate at the same site. At both 5' and 3', the frequency of a nucleotide at a given position in the zebrafish sequence is positively correlated with its conservation in chicken and mammals, suggesting that selection continued to operate in birds and mammals along similar lines.

Cyclooxygenase-2-derived prostacyclin protective role on endotoxin-induced mouse cardiomyocyte mortality.

Cardiovascular dysfunction characterizes septic shock, inducing multiple organ failure and a high mortality rate. In the heart, it has been shown an up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions with subsequent overproduction of nitric oxide (NO) and eicosanoids. This study is focused on the links between these products of inflammation and cell loss of mouse cardiomyocytes during treatment by the Salmonella typhimurium lipopolysaccharide (LPS) in presence or in absence of NOS or COX inhibitors. LPS induced RelA/NF-κB p65 activation, iNOS and COX-2 up-regulations, resulting in NO and prostacyclin releases. These effects were reversed by the NO-synthase inhibitor and increased by the specific COX-2 inhibitor. Immunostainings with FITC-conjugated anti-Annexin-V and propidium iodide and caspase 3/7 activity assay showed that cardiomyocyte necrosis was inhibited by L-NA during LPS treatment challenge, while apoptosis was induced in presence of both LPS and NS-398. No effect on LPS cellular injury was observed using the specific cyclooxygenase-1 (COX-1) inhibitor, SC-560. These findings strongly support the hypothesis of a link between iNOS-dependent NO overproduction and LPS-induced cell loss with a selective protective role allotted to COX-2 and deriving prostacyclins.

Plasma membrane damage sensing and repairing. Role of heterotrimeric G-proteins and the cytoskeleton.

Different toxic agents, derived from bacteria, viruses or cells of the immune system, as well as mechanical forces generated during cell locomotion are able to open pores in the cell plasma membrane. Most of these biological agents operate through specific receptors. We studied the formation and resealing of the "non-specific" plasma membrane pores generated by the mild non-ionic detergent Triton X-100. In HL-60-derived granulocytic cells plasma membrane pore opening after a 1-h treatment with Triton X-100 is documented by entry into the cell of the membrane impermeant dye ethidium bromide. As a consequence of the opening of pores the intracellular K(+) concentration falls dramatically, the cytosolic pH diminishes and the cell membrane is depolarized. Furthermore the cells acquire a polarized morphology, demonstrating the involvement of the actin cytoskeleton. At the Triton concentration used the membrane lesions are progressively repaired and by 8h the impermeability to ethidium bromide is restored and the intracellular K(+) concentration is virtually normal. Following treatments with Triton+Pertussis toxin, Triton+Cytochalasin, or Triton+Pertussis toxin+Cytochalasin the progress of membrane repair is dramatically slowed and is no longer completed by 8h. It is concluded that the membrane damage activates pertussis-sensitive G-proteins which likely act as sensors of the damage, while both G-proteins and the actin cytoskeleton are involved in the membrane repair mechanism.

MPTP-induced neuroinflammation increases the expression of pro-inflammatory cytokines and their receptors in mouse brain.

Parkinson's disease (PD) is a common neurodegenerative disease characterised by a slow and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Despite intensive research, the cause of neuronal loss in PD is poorly understood. Inflammatory mechanisms have been implicated in the pathophysiology of PD. In this study, conducted on an experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, we investigated the expression of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-6 and their receptors (IL-1RI, TNF-αRI, IL-6Rα) at the SN and caudate-putamen (CP) levels. In MPTP-treated animals we observed a significant increase in IL-1ß, TNF-α and IL-6 mRNA expression levels both in the SN and CP in comparison with untreated mice. In addition, both mRNA and protein levels of IL-1RI, TNF-αRI and IL-6Rα were significantly enhanced in the SN of MPTP-treated mice in comparison to controls, whereas no significant differences were observed in the CP between treated and untreated mice. Overall, these results indicate a role of both pro-inflammatory cytokines and their receptors in the pathogenesis of PD.

Lipopolysaccharide (LPS) of helicobacter modulates cellular DNA repair systems in intestinal cells.

The epithelium of the intestinal tract is exposed to a variety of genotoxic agents, both exogenous and endogenous, that can injure nuclear and mitochondrial DNA. DNA damage can be repaired by a series of DNA repair enzymes, while defects in this system will make these cells once more susceptible to malignant transformation or cell death. Recent studies suggest that intestinal bacteria may contribute to induce inflammation in individuals afflicted by inflammatory bowel disease (IBD), increasing the risk of developing colon cancer. Accumulating evidence suggests that Helicobacter organisms are linked to IBD as well as to gastric and colon cancer. Therefore, the focus of this study was to evaluate the effect of lipopolysaccharide (LPS) isolated from Helicobacter on modulating the DNA repair system. We used an in vitro model represented by two colon carcinoma cell lines, the DNA repair-proficient SW480 and the DNA repair-deficient LoVo, and transfected with a UVC-irradiated psV-beta-galactosidase plasmid. We observed that LPS, by upregulating the expression of inducible nitric oxide (NO), leads to an increased NO release, demonstrating that LPS is able to interfere with the DNA repair machinery of intestinal cells, thus increasing the risk of permanent genotoxic effects.

A new generation of MDR modulating agents with dual activity: P-gp inhibitor and iNOS inducer agents.

MultiDrug Resistance (MDR) is due to the ability of some ATPase transporters to efflux chemotherapeutic agents out from tumor cells decreasing the endocellular concentration for the pharmacological effect, causing cancer cells chemoresistance. In the present work, a set of MDR modulating agents (MC89, MC70, PB28, IG9) able to modulate transmembrane ATP-dependent transporter, P-glycoprotein (P-gp), and also to induce inducible nitric oxide synthase (iNOS) expression in a panel of tumor cell lines are presented. All selected compounds, known as potent P-gp modulating agents, stimulated nitric oxide (NO) via iNOS in U937, Caco-2 and MCF7-Adr cell lines. The results displayed a new pharmacological strategy to revert MDR and lead to develop a new class of MDR reverting agents devoid of the limits of P-gp inhibitors third generation.

First identification of Toll-like receptor-4 in avian brain: evolution of lipopolysaccharide recognition and inflammation-dependent responses.

In this work, we examine the effects of lipopolysaccharide (LPS) treatment on nerve cells of chick embryo used as a universal avian model. We demonstrate that LPS leads to a dramatic cell loss in primary cultures of both glia and neurons, isolated from chick embryos. Toxic effects appear to be mediated by the Toll-like receptor (TLR)-4 complex, expressed in both glial and neuronal cells, since after TLR-4 silencing by RNA interference experiments LPS-induced cytotoxicity was prevented. The role of nitric oxide in LPS-induced cell damage has also been investigated. These results demonstrate, for the first time in avian nerve cells, the surface expression of TLR-4 and its role as a pattern recognition receptor involved in LPS-induced cell responses in a similar manner to that observed in mammals.

Expression of pro-inflammatory TACE-TNF-α-amphiregulin axis in Sjögren's syndrome salivary glands.

The tumor-necrosis-factor-converting-enzyme (TACE)-TNF-α-Amphiregulin (AREG) axis plays an important pathogenic role in inflammatory and autoimmune disorders. However, the pathological roles of these proteins in the chronic autoimmune disease Sjögren's syndrome (SS) remain to be elucidated. It is known that the TACE-AREG axis is clearly part of a larger cascade of signals that starts with the activation of Furin, responsible for maturation of TACE that, in turn, determines the production of active TNF-α, directly involved in the up-regulation of AREG expression. This study showed that Furin, TACE, TNF-α, and AREG proteins, detected in acinar and ductal cells of human salivary glands from SS patients, increased remarkably in comparison with biopsies of labial salivary glands from healthy controls. The changes in Furin, TACE, TNF- α, and AREG proteins' level detected in salivary glands biopsies of SS patients could be responsible for pro-inflammatory cytokines overexpression characterizing Sjögren's syndrome.

RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

The aim of this study was to analyze the Furin-TNF-α-converting enzyme (TACE)-amphiregulin (AREG)-IL-6/IL-8 secretion pathway in non-neoplastic human salivary gland epithelial cells (SGECs) stimulated with anti-Ro/SSA autoantibodies (Abs). We examined whether anti-Ro/SSA Abs-mediated TACE activation is responsible for AREG activation. As recent studies have demonstrated that AREG could induce proinflammatory cytokines secretion in epithelial cells, we discuss how TACE-mediated AREG shedding, caused by anti-Ro/SSA Abs treatment, could have a critical role in TNF-α-induced IL-6 and IL-8 secretion by SGEC. Furthermore, the effects of TNF-α blockade on AREG expression and TNF-α-AREG-mediated IL-6 and IL-8 secretion were evaluated. We have discovered that the upregulation of AREG occurs through TNF-α produced after anti-Ro/SSA Abs uptake via Fcγ receptors. Biological drug adalimumab and the gene silencing technique were used to study the AREG-IL-6/IL-8 secretion pathway, demonstrating that (i) adalimumab-mediated TNF-α blocking and TNF-α gene silencing provoke a significant decrease of proinflammatory cytokines production and AREG expression in anti-Ro/SSA Abs-treated SGEC; (ii) AREG gene silencing has a potent inhibitory effect on TNF-α-induced IL-6 and IL-8 secretion in SGEC treated with anti-Ro/SSA Abs; (iii) an inspection of the kinetics of cytokine production after exogeni TNF-α and AREG addition, and the use of cycloheximide in the presence of exogenous TNF-α as stimulant, clarified that TNF-α induces IL-6 and IL-8 secretion through AREG.Laboratory Investigation advance online publication, 20 September 2010; doi:10.1038/labinvest.2010.168.

Pro-inflammatory role of Anti-Ro/SSA autoantibodies through the activation of Furin-TACE-amphiregulin axis.

Prolonged inflammation can be detrimental because it may cause host toxicity and tissue damage. Indeed, excessive production of inflammatory cytokines is often associated with many autoimmune diseases. In this study we demonstrate that the anti-Ro/SSA autoantibodies (Abs) stimulate the production of pro-inflammatory cytokines IL-6 and IL-8 by human healthy salivary gland epithelial cells (healthy SGEC). The secretion of these cytokines is due to amphiregulin (AREG) that is overexpressed in healthy SGEC treated with anti-Ro/SSA Abs and in Sjögren's syndrome. We have discovered that the up-regulation of AREG occurs through TNF-alpha produced following anti-Ro/SSA Abs treatment. The gene silencing technique was used to study the AREG-TNF-alpha-IL-6/IL-8 secretion pathway, demonstrating that: (i) TNF-alpha gene silencing provokes a significant decrease of proinflammatory cytokines production and AREG expression in anti-Ro/SSA Abs-treated healthy SGEC; (ii) AREG gene silencing has a potent inhibitory effect on TNF-alpha-induced IL-6 and IL-8 secretion in healthy SGEC treated with anti-Ro/SSA Abs. These findings indicate that TACE-mediated AREG shedding plays a critical role in TNF-alpha-induced IL-6 and IL-8 secretion by the human healthy salivary gland epithelial cells, suggesting that this may be one of the possible intracellular mechanisms involved in the salivary glands inflammatory response in Sjögren's syndrome.

Proposing a relationship between Mycobacterium avium subspecies paratuberculosis infection and Hashimoto's thyroiditis.

Humans are widely exposed to Mycobacterium avium subspecies paratuberculosis (MAP), a proven multi-host chronic enteric pathogen that has recently been linked to autoimmune diabetes. In the present study we used a MAP species-specific polymerase chain reaction with the insertion element IS900-specific probe to detect MAP infection in members of the same family suffering from Hashimoto's thyroiditis.

Inflammatory responses in embryonal cardiomyocytes exposed to LPS challenge: an in vitro model of deciphering the effects of LPS on the heart.

This study is focused on the links between the major products of inflammation and cell damage induced by the administration of lipopolysaccharide (LPS) from Salmonella typhimurium in embryonal cardiomyocytes. LPS treatment for 72 hours induced transcription factor NF-kappaB activation, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression, nitric oxide (NO) and tumor necrosis factor (TNF)-alpha release. Moreover, LPS administration induced a significant cell loss, reversed by the NO-synthases inhibitor, suggesting a relationship between cell damage and iNOS-dependent NO overproduction. Cell death was reversed by the specific NF-kappaB inhibitor, TPCK, whereas COX-2 specific inhibitor determined an increase of cell damage in terms of apoptosis, as observed by YO-PRO immunostaining, DNA laddering analysis and caspase-3 activation. Overall these findings evidenced a selective role for NF-kappaB in mediating NO-induced cell damage and a protective action by COX-2 in LPS-treated embryonal cardiomyocytes. The reflection of these experiments on human cardiac pathology will be discussed.

TNF blocker drugs modulate human TNF-α-converting enzyme pro-domain shedding induced by autoantibodies.

Novel biologic therapies targeted against specific components of the immune system, including blockade of TNF-α have revolutionized therapeutic approaches to inflammatory conditions and systemic inhibitors of TNF-α have been approved for the treatment of a wide variety of autoimmune diseases. No studies aimed to elucidate the effects of anti-TNF-α blockers on tumour necrosis factor-α convertase (TACE) expression and activation have yet been published. TACE is the principal protease involved in the activation of pro-TNF-α and is a target for anti-TNF-α therapy. Here we focused on regulation of TACE expression in human salivary gland epithelial cells (SGEC) treated by anti-Ro/SSA autoantibodies (autoAbs), characterizing primary Sjögren's syndrome and on the effect of anti-Ro/SSA autoAbs on TACE pro-domain shedding and activation. To test the hypothesis that anti-TNF-α blocker drugs affect TACE expression, we used Adalimumab and Etanercept to block TNF-α and evaluate the effects of these biological agents on post-translational regulation of TACE. Anti-Ro/SSA autoAbs determines TACE pro-domain shedding suggesting that TACE activity is necessary for the release of TNF-α observed in anti-Ro/SSA autoAbs-stimulated cells. The comparative efficacy analysis of the regulation of TACE activity by Adalimumab and Etanercept revealed that Adalimumab appear to be significantly more efficacious than Etanercept in preventing TACE activation caused by anti-Ro/SSA autoAbs. It is intriguing to consider that regulation of TACE may participate in the pathogenic role of autoantibodies and the modulation of TACE expression by TNF-α antagonists might contribute to the beneficial effect of these drugs in inflammatory and autoimmune diseases.

Regulation of mRNA caspase-8 levels by anti-nuclear autoantibodies.

Apoptosis of the acinar and ductal epithelial cells of the salivary glands has been proposed as a mechanism possibly responsible for the impairment of the secretory function in Sjögren's syndrome, an organ-specific autoimmune disorder characterized by destruction of these glandular structures. The presence of serum autoantibodies (Abs) directed against the ribonucleoproteic antigens Ro and La is one of the classification criteria used to identify Sjögren patients, and there is increasing evidence of the direct involvement of Abs in tissue pathogenesis. Our recent report demonstrated that anti-Ro and anti-La Abs are able to trigger the extrinsic pathway of apoptosis in the human salivary gland cells. To better understand how the anti-Ro and anti-La Abs exert their apoptotic effect, human caspase-8 gene expression was examined in primary human salivary gland epithelial cell (SGEC) cultures established from biopsies of labial minor salivary glands. To measure mRNA expression changes of initiating caspase-8, the real-time polymerase chain reaction was employed. This was combined with western blot to study the activation of caspase-8 detecting the cleaved form of caspase-8 and the cleaved poly (ADP-ribose) polymerase, downstream consequences of caspases activation. Data obtained suggest that the anti-Ro and anti-La Abs determine a transcriptional up-regulation and activation of caspase-8. Study of the mRNA in SGEC experimental model may provide insight into the signal transduction pathway stimulated by anti-nuclear autoantibodies.

Expression of UDP-glucuronosyltransferase 1A6 isoform in Caco-2 cells stimulated with lipopolysaccharide.

Glucuronidation is an important metabolic process of detoxification in all vertebrates. The reaction is catalyzed by a multigene family of UDP-glucuronosyltransferases (UGTs) able to convert many xenobiotics and endobiotics (hydrophobic substances) to inactive, water-soluble glucuronides. The UGTs play a protective role, facilitating the elimination of potentially toxic metabolites via urine, bile and feces; therefore, impairment of UGTs may have important toxicological consequences. The regulation of UGTs during bacterial infection or inflammation is not well described. In this study, we investigated the in vitro effect of lipopolysaccharide (LPS) on the expression of the UGT1A6 isoform in human colon carcinoma Caco-2 cells. Results demonstrated a significant down-regulation of UGT1A6 expression, both in terms of mRNA and protein levels, and a reduced UGT activity after LPS exposure of cell cultures, suggesting a role for endotoxins on UGT regulation mechanisms.

Induction of TNF-alpha-converting enzyme-ectodomain shedding by pathogenic autoantibodies.

The release of the soluble form of tumor necrosis factor (TNF)-alpha from the plasma membrane occurs through the activation of the secretase tumor necrosis factor-alpha-converting enzyme (TACE). The current study was designed to examine whether the anti-Ro/SSA autoantibodies (Abs) are capable to regulate TACE expression in non-neoplastic human salivary gland epithelial cells (SGEC) cultures. We investigated the effect of anti-Ro/SSA Abs on the localization and abundance of cell-surface TACE and on TACE pro-domain-shedding and activation. In addition, the potential physiological consequences of TNF-alpha blockage by the biological agent Adalimumab on post-translational regulation of TACE are discussed. Anti-Ro/SSA Abs were purified from IgG fractions of patients with primary Sjögren's syndrome, using Sepharose 4B-Ro/SSA affinity columns. Flow cytometry, reverse transcription-PCR, western blot and immunohistochemistry were used to study TACE expression on SGEC and TACE regulation by Abs. Our study demonstrated a dose-dependent increase of TACE messenger RNA (mRNA) expression in anti-Ro/SSA Abs-treated SGEC, followed by internalization, pro-domain shedding and activation of TACE protein, suggesting that increased TACE activity is necessary for the release of TNF-alpha observed in anti-Ro/SSA Abs-stimulated SGEC. Adalimumab treatment brought TACE mRNA and surface TACE expression to levels than those observed in untreated SGEC. These data suggest that the effect of anti-Ro/SSA Abs on TACE expression and intracellular distribution is exerted by TNF-alpha production.