Type 1–type 2 interferon imbalance in dry eye disease

Purpose: Dry eye disease (DED) is characterized by altered ocular surface proinflammatory and antiinflammatory factors. Interferons (IFNs) are a class of pleiotropic cytokines well known for their antimicrobial, inflammatory, and immunomodulatory roles. Hence, this study investigates the ocular surface expression of different types of IFNs in patients with DED. Methods: The cross?sectional, observational study included patients with DED and normal subjects. Conjunctival impression cytology (CIC) samples were obtained from the study subjects (controls, n = 7; DED, n = 8). The mRNA expression levels of type 1 IFN (IFN?, IFN?), type 2 IFN (IFN?), and type 3 IFN (IFN?1, IFN?2, IFN?3) were measured by quantitative PCR (polymerase chain reaction) in CIC samples. IFN? and IFN? expression under hyperosmotic stress was also studied in human corneal epithelial cells (HCECs) in vitro. Results: The mRNA expression levels of IFN? and IFN? were significantly lower and that of IFN? was significantly higher in DED patients compared to healthy controls. The mRNA levels of IFN?, IFN?, and IFN? were significantly lower compared to IFN? in DED patients. An inverse association between tonicity?responsive enhancer?binding protein (TonEBP; hyperosmotic stress maker) and IFN? or IFN? expression and a positive association between TonEBP and IFN? expression was observed in CIC samples. The expression of IFN? was lower than IFN? in HCECs undergoing hyperosmotic stress compared to HCECs without the stress. Conclusion: The presence of an imbalance between type 1 and type 2 IFNs in DED patients suggests newer pathogenic processes in DED, plausible ocular surface infection susceptibility in DED patients, and potential therapeutic targets in the management of DED

TonEBP suppresses adipocyte differentiation via modulation of early signaling in 3T3-L1 cells

TonEBP belongs to the Rel family of transcription factors and plays important roles in inflammation as well as kidney homeostasis. Recent studies suggest that TonEBP expression is also involved in differentiation of several cell types such as myocytes, chondrocytes, and osteocytes. In this study, we investigated the roles of TonEBP during adipocyte differentiation in 3T3-L1 cells. TonEBP mRNA and protein expression was dramatically reduced during adipocyte differentiation. Sustained expression of TonEBP using an adenovirus suppressed the formation of lipid droplets as well as the expression of FABP4, a marker of differentiated adipocytes. TonEBP also inhibited the expression of PPARγ, a known master regulator of adipocytes. RNAi-mediated knock down of TonEBP promoted adipocyte differentiation. However, overexpression of TonEBP did not affect adipogenesis after the initiation of differentiation. Furthermore, TonEBP expression suppressed mitotic clonal expansion and insulin signaling, which are required early for adipocyte differentiation of 3T3-L1 cells. These results suggest that TonEBP may be an important regulatory factor in the early phase of adipocyte differentiation.

Expression of TonEBP by Hypertonic and Hyperosmolar Stress in RGC-5 Cells

PURPOSE: In order to determine whether the Tonicity responsive enhancer binding protein (TonEBP) is expressed by hypertonic and hyperosmolar stress, TonEBP expression was investigated in the retinal ganglion cell (RGC) line, RGC-5 cells. METHODS: After RGC-5 cells were cultured by Staurosporine, TonEBP expression was measured with Western immunoblotting analysis and real-time reverse transcription-polymerase chain reaction in 50 mM NaCl, 100 mM mannitol, 50 mM glucose, or 100 mM glucose at 3, 6, 12, and 24 hours after exposure to each environment. RESULTS: In this study, the protein expression of TonEBP was determined to be statistically significantly checked in 50 mM NaCl after 3, and 6 hours, in 100 mM mannitol after 6 hours, and in 100 mM glucose after 3, and 6 hours. TonEBP messenger Ribonucleic acid (mRNA) expression was determined to be statistically significantly checked in 50 mM NaCl after 3 hours, in 100 mM mannitol after 3, and 24 hours, and in 50 mM glucose after 3, and 24 hours. CONCLUSIONS: These results suggested that TonEBP was expressed by hypertonic and hyperosmolar stress at the protein and mRNA levels. Further studies are nedded to determine the role of TonEBP and the mechanism of expression and regulation of TonEBP.

TonEBP and SMIT expression in human placenta / 대한해부학회지

Tonicity-responsive enhancer binding protein (TonEBP) is a signal transcription factor of transporters such as sodium-myo-inositol cotransporter (SMIT), aldose reductase. TonEBP has a variety of functions such as control of intracellular osmolytes and immunomodulating. It is known that TonEBP is abundant in the placenta, but location and function aren't known. The aim of this study is to describe the localization of TonEBP in the placenta. We assayed the immunohistochemistry of TonEBP and performed in situ hybridization of SMIT in normal human full term placenta. In normal human full term placenta, TonEBP was in villous trophoblasts, extravillous trophoblasts and some endothelial cells. The result of the in situ hybridization of SMIT was similar to that of immunohistochemistry of TonEBP. Neither TonEBP nor SMIT was present in TonEBP knockout mouse placenta. This shows TonEBP is a key factor in SMIT transcription. TonEBP may play an important role in transporting of inositol to fetus in placenta.

Hyperosmotic Stimulus Down-regulates 1alpha, 25-dihydroxyvitamin D3-induced Osteoclastogenesis by Suppressing the RANKL Expression in a Co-culture System

The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on 1alpha,25(OH)2D3-induced osteoclastogenesis was investigated in an osteoblast-preosteoclast co-culture system. Serial doses of sucrose were applied as a mechanical force. These hyperosmotic stimuli significantly evoked a reduced number of 1alpha,25(OH)2D3-induced tartrate-resistant acid phosphatase-positive multinucleated cells and 1alpha,25(OH)2D3-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor kappaB ligand (RANKL) and Runx2 expressions were down-regulated in response to 1alpha,25(OH)2D3. Knockdown of Runx2 inhibited 1alpha,25(OH)2D3-induced RANKL expression in osteoblastic cells. Finally, the hyperosmotic stimulus induced the overexpression of TonEBP in osteoblastic cells. These results suggest that hyperosmolarity leads to the down-regulation of 1alpha,25(OH)2D3-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.

Urine Concentration and the Adaptation of Renal Medullary Cells to Hypertonicity

Hypertonicity(hypernatremia) of extracellular fluid causes water movement out of cells, while hypotonicity(hyponatremia) causes water movement into cells, resulting in cellular shrinkage or cellular swelling, respectively. In most part of the body, the osmolality of extracellular fluid is maintained within narrow range(285-295 mOsm/kgH2O) and some deviations from this range are not problematic in most tissue of the body except brain. On the other hand, the osmolality in the human renal medulla fluctuates between 50 and 1,200 mOsm/kgH2O in the process of urine dilution and concentration. The adaptation of renal medullary cells to the wide fluctuations in extracellular tonicity is crucial for the cell survival. This review will summarize the mechanisms of urine concentration and the adaptation of renal medullary cells to the hypertonicity, which is mediated by TonEBP transcription factor and its target gene products(UT- A1 urea transporter etc.).

Alternative Isoforms of TonEBP with Variable N-termini are Expressed in Mammalian Cells

Hypertonicity imposes a great deal of stress to cells since it causes rise in cellular ionic strength, which can be reduced by the accumulation of compatible osmolytes. TonEBP plays a central role in the cellular accumulation of compatible osmolytes via transcriptional stimulation of membrane transporters and aldose reductase. Alternatively spliced forms of TonEBP mRNA have previously been reported and two of them showed different transcriptional activity. In the present study, isoform-specific antibodies were produced to confirm the translation of the spliced mRNA to protein. TonEBP was immunoprecipitated by using anti-TonEBP antibody and then immunoblotted using anti-TonEBP or isoform specific antibodies to find out the expression profile of TonEBP isoforms in basal or stimulated condition. From these results, we conclude that all TonEBP isoforms are expressed in mammalian cells and their expression patterns are not same in every cells.

The Abundance and Nuclear Distribution of TonEBP in Response to Changes of Tonicity in Hyperglycemic Cells / 대한내분비학회지

BACKGROUND: TonEBP (Tonicity-responsive enhancer binding protein) regulates the transcription of tonicity responsive genes, such as sodium-myo-inositol and the sodium- chloride-betaine co- transporters (SMIT & BGT1), heat shock protein 70 (HSP70) and aldose reductase (AR). To characterize the signals that activate TonEBP in hyperglycemic human retinal pigment epithelial (hRPE) cells, the abundance and nuclear distribution of TonEBP were studied in response to changes of tonicity in culture media. METHODS: After the cultures reached confluence, the hRPE cells were exposed for 3 days to 25 mM glucose and 100 mM NaCl, both with and without 20 M tolrestat. The expressions of AR, SMIT and HSP 70 were determined by northern blot, and the abundances of TonEBP by western blot. The nuclear distributions of TonEBP were observed by fluorescence microscopy, after immuno staining. RESULTS: The AR and SMIT mRNA levels in hyperglycemic and hypertonic media were decreased compared to those in hypertonic media alone. These decreased AR and SMIT mRNA expressions were also observed to be significantly prevented in those cells incubated with tolrestat. Stimulation of TonEBP in hypertonic medium occurs due to a combination of an increased abundance of TonEBP and an increased distribution into the nucleus from the cytoplasm. However, the expressions and nuclear distributions of TonEBP in hyperglycemic and hypertonic media were not different from those in hypertonic media alone. CONCLUSION: The expressions of AR and SMIT genes that may influence the development of diabetic complication were down-regulated by the intracellular accumulation of sorbitol in sustained hyperglycemia. TonEBP does not play a key role in the hypertonicity-induced transcriptional regulation of AR and SMIT in hyperglycemic cells, due to the intracellular accumulation of sorbitol and depletion of myo-inositol

Expression of Tonicity-Responsive Enhancer Binding Protein (TonEBP) in the Rat Cochlea: An Immunohistochemical Study / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The inner ear is an organ used for hearing and balance. For its normal function, the inner ear fluid homeostasis is required. There has been controversy over the regulatory mechanisms of maintaining inner ear fluid balance, and they have not yet been clearly defined. TonEBP is the protein that binds tonicity-responsive enhancer elements in the osmoprotective gene, which elevates the compatible osmolytes, which in turn induces cell survival in hypertonic condition. The aim of this study was to elucidate if there is an osmoregulatory mechanism in cochlea. Material and Method: The localization of TonEBP in the cochlea of male Sprague-Dawley rats was studied by immunohistochemistry with an anti rabbit polyclonal anti-rat TonEBP antibody. RESULTS: TonEBP was expressed at outer hair cells, Deiter cells, spiral ligaments, sprial limbus connective tissues, and epithelial lining of basilar membrane facing scala tympani. CONCLUSION: TonEBP in cochlea is one of the proteins involved in elucidating cell survival in changed tonicity during inner ear homeostasis.