An improved strategy of TGFß3 expression in Escherichia coli: Exploiting folding modulators for a switch from misfolded to folded form.

Transforming growth factor beta 3 (TGFß3) exhibits a complex native structure featuring the presence of multiple disulfide bonds forming the active dimer. Consequently, its heterologous expression in microbial system invariably leads to inclusion body (IB) formation. In this study, we observed an interesting phenomenon of switching a significant fraction of misfolded TGFß3 to folded form by modulating the cellular protein folding machinery. We carried out co-expression experiments with chaperones and demonstrated the requirement of a coordinated action of DnaK-DnaJ-GrpE and GroESL, to achieve the native soluble conformation of TGFß3, during over-expression in E. coli. The novelty of this study lies in the fact that orchestration of a group of chaperones to work in concert for efficient folding and assembly of TGFß3-like cytokines has not been widely explored. Additionally, we have also demonstrated that presence of osmolytes (sorbitol or trehalose) in the growth media have an appreciable impact on the solubility of TGFß3. We have further shown a synergism between the effects of molecular chaperone and osmolytes on the solubility of TGFß3. We have confirmed the functionality of soluble TGFß3 by performing binding interactions with its cognate receptor TßRII. Our study delineates the fact that an effective combination of chaperones or optimum concentration of compatible osmolyte, can efficiently abrogate competing aggregation pathways and help attain the native conformation of a cysteine rich cytokine in a facile manner.

The autophagy protein Ambra1 regulates gene expression by supporting novel transcriptional complexes.

Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology.

Therapeutic Effects of Focused Ultrasound on Expression of Notch1, C-Fos and Transforming Growth Factor-ß3 in Vulvar Skin of SD Rats with Vulvar Lichen Simplex Chronicus.

The purpose of this study was to investigate the therapeutic effects of focused ultrasound on the expression of notch1, c-fos and transforming growth factor-ß3 (TGF-ß3) in genital skin of SD rats with vulvar lichen simplex chronicus (LSC). Fifty-six female SD rats with LSC were randomly divided into therapy and sham groups. The therapy group was exposed to focused ultrasound. The sham group received the same therapy with an instrument that had no power output. Four wk after a singly focused ultrasound therapy, histologic analyses revealed that recovered SD rats accounted for 75% of SD rats in the therapy group and 10.7% in the sham group. Total collagen fiber density in the superficial layer of dermis in the therapy group was significantly lower than that in the sham group. Notch1 and c-fos protein expression in the therapy group was significantly lower than that in the sham group, with the opposite effect present for TGF-ß3. Focused ultrasound therapy may inhibit superficial collagen fibrosis in the dermis by affecting expression of notch1, c-fos and TGF-ß3 in vulvar skin tissue and consequently reduce the recurrence rate of LSC.

Effect of TGFß1, TGFß3 and keratinocyte conditioned media on functional characteristics of dermal fibroblasts derived from reparative (Balb/c) and regenerative (Foxn1 deficient; nude) mouse models.

Skin injuries in mammals are healed through repair or regeneration. Our previous studies demonstrated that deficient expression of the transcription factor Foxn1 in epidermis of nude mice accounts for their skin's pronounced regenerative properties. Since homeostasis within the skin depends on complex interactions between the epidermal and underlying dermal layers, the present study characterizes and compares isolated dermal fibroblasts (DFs) between regenerative nude (Foxn1 deficient) mice and their wild-type Balb/c counterparts. Nude DFs exhibited a higher cumulative number of population doublings (cumulative PD) at low seeding density and increased adipogenic differentiation capacity relative to their Balb/c DF counterparts. Nude DFs displayed reduced migration and gel contraction, functional features associated with wound healing. The comparison of transforming growth factor ß family (TGFß) expression showed significantly higher levels of Tgfß3 transcript between nude and Balb/c mice but no differences were detected for Tgfß1. Nude DFs were specifically sensitive to the presence of the pro-regenerative TGFß3 isoform, showing increased collagen I deposition and alpha smooth muscle actin expression. Viability of Balb/c DFs was stimulated by keratinocyte conditioned media (KCM) from Balb/c (Foxn1 active) but inhibited by nude (Foxn1 deficient) KCM. In contrast, nude DFs did not respond to either KCMs with respect to their metabolic activity. Collectively, the enhanced plasticity and greater sensitivity of nude DFs to TGFß3 stimulation are indicative of and consistent with their pro-regenerative characteristics. These data support the hypothesis that epidermal Foxn1 plays a critical role in determining the DFs regenerative phenotype.

Effect of tamoxifen on fibrosis, collagen content and transforming growth factor-ß1, -ß2 and -ß3 expression in common bile duct anastomosis of pigs.

End-to-end anastomosis in the treatment for bile duct injury during laparoscopic cholecystectomy has been associated with stricture formation. The aim of this study was to experimentally investigate the effect of oral tamoxifen (tmx) treatment on fibrosis, collagen content and transforming growth factor-ß1, -ß2 and -ß3 expression in common bile duct anastomosis of pigs. Twenty-six pigs were divided into three groups [sham (n = 8), control (n = 9) and tmx (n = 9)]. The common bile ducts were transected and anastomosed in the control and tmx groups. Tmx (40 mg/day) was administered orally to the tmx group, and the animals were euthanized after 60 days. Fibrosis was analysed by Masson's trichrome staining. Picrosirius red was used to quantify the total collagen content and collagen type I/III ratio. mRNA expression of transforming growth factor (TGF)-ß1, -ß2 and -ß3 was quantified using real-time polymerase chain reaction (qRT-PCR). The control and study groups exhibited higher fibrosis than the sham group, and the study group showed lower fibrosis than the control group (P = 0.011). The control and tmx groups had higher total collagen content than the sham group (P = 0.003). The collagen type I/III ratio was higher in the control group than in the sham and tmx groups (P = 0.015). There were no significant differences in the mRNA expression of TGF-ß1, -ß2 and -ß3 among the groups (P > 0.05). Tmx decreased fibrosis and prevented the change in collagen type I/III ratio caused by the procedure.

Egr2 and Egr3 in regulatory T cells cooperatively control systemic autoimmunity through Ltbp3-mediated TGF-ß3 production.

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by multiorgan inflammation induced by autoantibodies. Early growth response gene 2 (Egr2), a transcription factor essential for T-cell anergy induction, controls systemic autoimmunity in mice and humans. We have previously identified a subpopulation of CD4+ regulatory T cells, CD4+CD25-LAG3+ cells, that characteristically express both Egr2 and LAG3 and control mice model of lupus via TGF-ß3 production. However, due to the mild phenotype of lymphocyte-specific Egr2-deficient mice, the presence of an additional regulator has been speculated. Here, we show that Egr2 and Egr3 expressed in T cells cooperatively prevent humoral immune responses by supporting TGF-ß3 secretion. T cell-specific Egr2/Egr3 double-deficient (Egr2/3DKO) mice spontaneously developed an early onset lupus-like disease that was more severe than in T cell-specific Egr2-deficient mice. In accordance with the observation that CD4+CD25-LAG3+ cells from Egr2/3DKO mice completely lost the capacity to produce TGF-ß3, the excessive germinal center reaction in Egr2/3DKO mice was suppressed by the adoptive transfer of WT CD4+CD25-LAG3+ cells or treatment with a TGF-ß3-expressing vector. Intriguingly, latent TGF-ß binding protein (Ltbp)3 expression maintained by Egr2 and Egr3 was required for TGF-ß3 production from CD4+CD25-LAG3+ cells. Because Egr2 and Egr3 did not demonstrate cell intrinsic suppression of the development of follicular helper T cells, Egr2- and Egr3-dependent TGF-ß3 production by CD4+CD25-LAG3+ cells is critical for controlling excessive B-cell responses. The unique attributes of Egr2/Egr3 in T cells may provide an opportunity for developing novel therapeutics for autoantibody-mediated diseases including SLE.

Expression signature of lncRNAs and their potential roles in cardiac fibrosis of post-infarct mice.

The present study aimed to investigate whether long non-coding RNAs (lncRNAs) are involved in cardiac fibrogenesis induced by myocardial infarction (MI). The differentially expressed lncRNAs and mRNAs in peri-infarct region of mice 4 weeks after MI were selected for bioinformatic analysis including gene ontology (GO) enrichment, pathway and network analysis. Left ventricular tissue levels of lncRNAs and mRNAs were compared between MI and sham control mice, using a false discovery rate (FDR) of <5%. Out of 55000 lncRNAs detected, 263 were significantly up-regulated and 282 down-regulated. Out of 23000 mRNAs detected, 142 were significantly up-regulated and 67 down-regulated. Among the differentially expressed lncRNAs, 53 were up-regulated by ≥2.0-fold change and 37 down-regulated by ≤0.5-fold change. Nine up-regulated and five down-regulated lncRNAs were randomly selected for quantitative real-time PCR (qRT-PCR) verification. GO and pathway analyses revealed 173 correlated lncRNA-mRNA pairs for 57 differentially expressed lncRNAs and 20 differentially expressed genes which are related to the development of cardiac fibrosis. We identified TGF-ß3 as the top-ranked gene, a critical component of the transforming growth factor-ß (TGF-ß) and mitogen activated protein kinase (MAPK) signalling pathways in cardiac fibrosis. NONMMUT022554 was identified as the top-ranked lncRNA, positively correlated with six up-regulated genes, which are involved in the extracellular matrix (ECM)-receptor interactions and the phosphoinositid-3 kinase/protein kinase B (PI3K-Akt) signalling pathway. Our study has identified the expression signature of lncRNAs in cardiac fibrosis induced by MI and unravelled the possible involvement of the deregulated lncRNAs in cardiac fibrosis and the associated pathological processes.

Gene Delivery of TGF-ß3 and BMP2 in an MSC-Laden Alginate Hydrogel for Articular Cartilage and Endochondral Bone Tissue Engineering.

Incorporating therapeutic genes into three-dimensional biomaterials is a promising strategy for enhancing tissue regeneration. Alginate hydrogels have been extensively investigated for cartilage and bone tissue engineering, including as carriers of transfected cells to sites of injury, making them an ideal gene delivery platform for cartilage and osteochondral tissue engineering. The objective of this study was to develop gene-activated alginate hydrogels capable of supporting nanohydroxyapatite (nHA)-mediated nonviral gene transfer to control the phenotype of mesenchymal stem cells (MSCs) for either cartilage or endochondral bone tissue engineering. To produce these gene-activated constructs, MSCs and nHA complexed with plasmid DNA (pDNA) encoding for transforming growth factor-beta 3 (pTGF-ß3), bone morphogenetic protein 2 (pBMP2), or a combination of both (pTGF-ß3-pBMP2) were encapsulated into alginate hydrogels. Initial analysis using reporter genes showed effective gene delivery and sustained overexpression of the transgenes were achieved. Confocal microscopy demonstrated that complexing the plasmid with nHA before hydrogel encapsulation led to transport of the plasmid into the nucleus of MSCs, which did not happen with naked pDNA. Gene delivery of TGF-ß3 and BMP2 and subsequent cell-mediated expression of these therapeutic genes resulted in a significant increase in sulfated glycosaminoglycan and collagen production, particularly in the pTGF-ß3-pBMP2 codelivery group in comparison to the delivery of either pTGF-ß3 or pBMP2 in isolation. In addition, stronger staining for collagen type II deposition was observed in the pTGF-ß3-pBMP2 codelivery group. In contrast, greater levels of calcium deposition were observed in the pTGF-ß3- and pBMP2-only groups compared to codelivery, with a strong staining for collagen type X deposition, suggesting these constructs were supporting MSC hypertrophy and progression along an endochondral pathway. Together, these results suggest that the developed gene-activated alginate hydrogels were able to support transfection of encapsulated MSCs and directed their phenotype toward either a chondrogenic or an osteogenic phenotype depending on whether TGF-ß3 and BMP2 were delivered in combination or isolation.

Survivin-TGFB3-TIMP1 Gene Therapy Via Lentivirus Vector Slows the Course of Intervertebral Disc Degeneration in an In Vivo Rabbit Model.

STUDY DESIGN: The ability of lentivirus vector (LV) survivin-transforming growth factor beta 3 (TGFB3)-tissue inhibitor of metalloproteinases 1 (TIMP1) on slowing disc degeneration was evaluated by an animal experiment. OBJECTIVE: The aim of the study was to investigate the effect of LV survivin-TGFB3-TIMP1 on slowing disc degeneration in an in vivo rabbit model. SUMMARY OF BACKGROUND DATA: Cell apoptosis, increase of catabolic activity, and decrease of anabolic activity were the mechanisms of disc degeneration. Meanwhile, survivin, TGFB3, and TIMP1 can influence above process, respectively. However, there were no researches conducted to evaluate the effect of an LV containing all three proteins (referred to as LV-survivin-TGFB3-TIMP1) on slowing disc degeneration in vivo. METHODS: Twenty skeletally mature female New Zealand White rabbits were randomly divided into four groups: nonpunctured sham surgical group (group A, n = 5), punctured blank control group (group B, n = 5), punctured empty vector control group (group C, n = 5), and the treatment group (group D, n = 5). Computed tomography-guided puncture was performed at the L3-L4 and L4-L5 discs, in accordance with a previously validated rabbit annulotomy model for intervertebral disc degeneration. After 3 weeks, LV-carrying survivin, TGFB3, and TIMP1 were injected into the nucleus pulposus. Serial magnetic resonance imaging studies at 0, 3, and 12 weeks were performed. The rabbits were sacrificed at 12 weeks, and the histology, immunofluorescence, quantitative real-time polymerase chain reaction, Western blot, and caspase-3 activity was used for evaluation. RESULTS: Magnetic resonance imaging, histology, gene expression, protein content, and apoptosis analyses of group A showed no disc degeneration. Groups B and C showed disc degeneration, which increased over time, and no significant difference was observed between the two groups (P > 0.05). In group D, there was less disc degeneration compared to the punctured control groups and the difference was statistically significant (P < 0.05). CONCLUSION: The injection of LV-carrying survivin-TGFB3-TIMP1 into punctured rabbit intervertebral discs helps delay degenerative disc changes. Although data from animal models should be extrapolated to the human condition with caution, this study shows promise for gene therapy to decelerate disc degeneration. LEVEL OF EVIDENCE: N/A.

Mesenchymal stem cells suppress fibroblast proliferation and reduce skin fibrosis through a TGF-ß3-dependent activation.

Recent studies showed that transplantation of mesenchymal stem cells (MSCs) significantly decreased tissue fibrosis; however, little attention has been paid to its efficacy on attenuating skin fibrosis, and the mechanism involved in its effect is poorly understood. In this work, we investigated the effects of MSCs on keloid fibroblasts and extracellular matrix deposition through paracrine actions and whether the antifibrotic properties of MSCs involved transforming growth factor-ß (TGF-ß)-dependent activation. In vitro experiments showed that conditioned media (CM) from MSCs decreased viability, a-smooth muscle actin expression, and collagen secretion of human keloid fibroblasts. In addition, TGF-ß3 secreted by MSCs was expressed at high level under inflammatory environment, and blocking the activity of TGF-ß3 apparently antagonized the suppressive activity of MSC CM, which demonstrated that TGF-ß3 played a preponderant role in preventing collagen accumulation. In vivo studies showed that MSC CM infusion in a mouse dermal fibrosis model induced a significant decrease in skin fibrosis. Histological examination of tissue sections and immunohistochemical analysis for α-smooth muscle actin revealed that TGF-ß3 of CM-mediated therapeutic effects could obviously attenuate matrix production and myofibroblast proliferation and differentiation. These findings suggest that TGF-ß3 mediates the attenuating effect of MSCs on both the proliferation and extracellular matrix production of human keloid fibroblasts and decreases skin fibrosis of mouse model, thus providing new understanding and MSC-based therapeutic strategy for cutaneous scar treatment.

Transforming growth factor-ß3 regulates cell junction restructuring via MAPK-mediated mRNA destabilization and Smad-dependent protein degradation of junctional adhesion molecule B (JAM-B).

Junctional adhesion molecule-B (JAM-B) is found between Sertoli cells at the blood-testis barrier (BTB) as well as between Sertoli and germ cells at the apical ectoplasmic specializations (ES) in the testis. The expression of JAM-B is tightly regulated to modulate the passage of spermatocytes across the BTB as well as the release of mature spermatozoa from the seminiferous epithelium. Transforming growth factor beta (TGF-ß) family is implicated in the regulation of testicular cell junction dynamics during spermatogenesis. This study aims to investigate the effects of TGF-ß3 on the expression of JAM-B as well as the underlying mechanisms on how TGF-ß3 regulates JAM-B expression to facilitate the disassembly of the BTB and apical ES. Our results revealed that TGF-ß3 suppresses JAM-B at post-transcriptional and post-translational levels. Inhibitor, siRNA knockdown and co-immunoprecipitation have shown that TGF-ß3 induces JAM-B protein degradation via ubiquitin-proteasome pathway. Immunofluorescence staining further confirmed that blockage of ubiquitin-proteasome pathway could abrogate TGF-ß3-induced loss of JAM-B at the cell-cell interface. siRNA knockdown and immunofluorescence staining also demonstrated that activation of Smad signaling is required for TGF-ß3-induced JAM-B protein degradation. In addition, TGF-ß3 reduces JAM-B mRNA levels, at least in part, via post-transcriptional regulation. mRNA stability assay has confirmed that TGF-ß3 promotes the degradation of JAM-B transcript and TGF-ß3-mediated mRNA destabilization requires the activation of ERK1/2 and p54 JNK signal cascades. Taken together, TGF-ß3 significantly downregulates JAM-B expression via post-transcriptional and post-translational modulation and results in the disruption of BTB and apical ES.

The perivascular niche governs an autoregulatory network to support breast cancer metastasis.

Unravelling the autoregulatory network that induces and maintains cancer stem cell state may provide novel effective therapies against breast cancer metastasis. The perivascular niche develops elements that initiate the autoregulatory machine to induce and maintain cancer stem cells, but not EMT, among newly arrived tumour cells. Inhibition of one or more primary key elements that trigger this circuit may result in the prevention or cure of breast cancer metastasis.

Genomic identification and molecular characterization of a non-mammalian TNFAIP8L2 gene from Oplegnathus fasciatus.

Tumor necrosis factor alpha-induced protein 8-like 2 (TNFAIP8L2) is a newly described negative immune regulator, whose enigmatic biological functions are not clearly understood. In the present study, the TNFAIP8L2 homolog of rock bream (Oplegnathus fasciatus) was identified and characterized. The genomic composition of rock bream TNFAIP8L2 (~6.7 kb) represented a tripartite arrangement in which three exons are interrupted by two introns. The rock bream TNFAIP8L2 transcript (1974 bp) possessed a coding sequence of 561 bp encoding a peptide of 186 amino acids. The predicted rock bream TNFAIP8L2 protein was 21.1kDa and revealed the typical features of known TNFAIP8L2 members including the DED-like domain. Rock bream TNFAIP8L2 was composed of six α-helices and demonstrated a distinct folding pattern of the TNFAIP8L2 family. It showed a certain degree of homology and phylogenetic relationship with the corresponding tilapia counterpart. Based on an interspecies genomic organizational comparison of TNFAIP8L2 orthologs, they could be classified into two classes, with teleost and non-teleost origin respectively. While teleost TNFAIP8L2s manifest a tripartite arrangement, non-teleost counterparts demonstrate a dipartite structure suggesting the loss of an intron during the post-piscine speciation. Promoter proximal region of rock bream TNFAIP8L2 consisted of multiple immune responsive cis-regulatory elements. Analysis of basal transcription in eleven tissues revealed its constitutive expression in examined tissues with highest magnitude in the head kidney. The modulated temporal mRNA expression of rock bream TNFAIP8L2 in head kidney post-challenges with stimulants (LPS and poly I:C) and pathogens (Streptococcus iniae and irido virus) was stimulant-specific. Additionally, a drastic down-regulation of rock bream TNFAIP8L2 mRNA level occurred in blood cells collected from experimentally injured animals, and it was accompanied by a contemporaneous down-regulation of cytokines, TNF-α and TGFß3. All these findings imply that rock bream TNFAIP8L2 is potentially responsible for immune and inflammatory modulation in rock bream.

Transforming growth factor-ß (TGF-ß) pathway abnormalities in tenascin-X deficiency associated with CAH-X syndrome.

Patients with congenital adrenal hyperplasia (CAH) with tenascin-X deficiency (CAH-X syndrome) have both endocrine imbalances and characteristic Ehlers Danlos syndrome phenotypes. Unlike other subtypes, tenascin-X-related Ehlers Danlos syndrome is caused by an extracellular matrix protein deficiency rather than a defect in fibrillar collagen or a collagen-modifying enzyme, and the understanding of the disease mechanisms is limited. We hypothesized that transforming growth factor-ß pathway dysregulation may, in part, be responsible for connective tissue phenotypes observed in CAH-X, due to this pathway's known role in connective tissue disorders. Fibroblasts and direct tissue from human skin biopsies from CAH-X probands and age- and sex-matched controls were screened for transforming growth factor-ß biomarkers known to be dysregulated in other hereditary disorders of connective tissue. In CAH-X fibroblast lines and dermal tissue, pSmad1/5/8 was significantly upregulated compared to controls, suggesting involvement of the bone morphogenetic protein pathway. Additionally, CAH-X samples compared to controls exhibited significant increases in fibroblast-secreted TGF-ß3, a cytokine important in secondary palatal development, and in plasma TGF-ß2, a cytokine involved in cardiac function and development, as well as palatogenesis. Finally, MMP-13, a matrix metalloproteinase important in secondary palate formation and tissue remodeling, had significantly increased mRNA and protein expression in CAH-X fibroblasts and direct tissue. Collectively, these results demonstrate that patients with CAH-X syndrome exhibit increased expression of several transforming growth factor-ß biomarkers and provide a novel link between this signaling pathway and the connective tissue dysplasia phenotypes associated with tenascin-X deficiency.

Topical photodynamic therapy following excisional wounding of human skin increases production of transforming growth factor-ß3 and matrix metalloproteinases 1 and 9, with associated improvement in dermal matrix organization.

BACKGROUND: Animal studies report photodynamic therapy (PDT) to improve healing of excisional wounds; the mechanism is uncertain and equivalent human studies are lacking. OBJECTIVES: To explore the impact of methyl aminolaevulinate (MAL)-PDT on clinical and microscopic parameters of human cutaneous excisional wound healing, examining potential modulation through production of transforming growth factor (TGF)-ß isoforms. METHODS: In 27 healthy older men (60-77 years), a 4-mm punch biopsy wound was created in skin of the upper inner arm and treated with MAL-PDT three times over 5 days. An identical control wound to the contralateral arm was untreated and both wounds left to heal by secondary intention. Wounds were re-excised during the inflammatory phase (7 days, n = 10), matrix remodelling (3 weeks, n = 8) and cosmetic outcome/dermal structure (9 months, n = 9). Production of TGF-ß1, TGF-ß3 and matrix metalloproteinases (MMPs) was assessed by immunohistochemistry alongside microscopic measurement of wound size/area and clinical assessment of wound appearance. RESULTS: MAL-PDT delayed re-epithelialization at 7 days, associated with increased inflammation. However, 3 weeks postwounding, treated wounds were smaller with higher production of MMP-1 (P = 0·01), MMP-9 (P = 0·04) and TGF-ß3 (P = 0·03). TGF-ß1 was lower than control at 7 days and higher at 3 weeks (both P = 0·03). At 9 months, MAL-PDT-treated wounds showed greater, more ordered deposition of collagen I, collagen III and elastin (all P < 0·05). CONCLUSIONS: MAL-PDT increases MMP-1, MMP-9 and TGF-ß3 production during matrix remodelling, ultimately producing scars with improved dermal matrix architecture.

Analysis of expression of TNF-alpha and TGF-beta3 in intrinsic ureteropelvic junction obstruction.

OBJECTIVES: Ureteropelvic junction (UPJ) obstruction is of critical importance to understand the histopathology of UPJ obstruction in terms of therapy planning and follow-up. For this purpose, our study was conducted with TNF-α and TGF-ß markers to investigate possible underlying problems in intrinsic UPJ obstruction. METHODS: Of the patients who had undergone surgery in our clinic, 36 UPJ segments of patients who had undergone dismembered pyeloplasty surgery due to UPJ obstruction and 14 UPJ segments of the patients who had undergone nephrectomy were collected to form 2 groups. All histological sections were examined by applying immunohistochemical transforming growth factor beta 3 (TGF-ß3) and tumour necrosis factor alpha (TNF-α) monoclonal antibody dyes. RESULTS: The mean staining values for TNF-α in mucosal tissue and mucosa were 0.53±0.84 and 0.58±0.84, respectively in the obstruction group, whereas the values observed in the control group were 0.86±0.36 and 0.93±0.47, respectively. While the mean staining values in the obstruction group in mucosal tissue and mucosa for TGF-ß3 were 1.75±0.73 and 2.17±0.77, respectively, the values established in the control group were 1.14±0.66 and 1.43±0.93, respectively. The difference between the obstruction and control groups were statistically significant for both values (p<0.05). CONCLUSION: Only a limited number of studies have been carried out on this particular issue. Data from the present study indicate that TGF-ß3 and TNF-α may play a role in the histopathogenesis of UPJ obstruction (Tab. 1, Fig. 1, Ref. 18).

Effects of electromagnetic radiation on morphology and TGF-ß3 expression in mouse testicular tissue.

Exposure to electromagnetic pulses in certain doses may lead to increase in the permeability of the blood testes barrier (BTB) in mice, which in turn affects spermatogenesis, penetration and spermiation. TGF-ß3 is a key molecule involved in BTB permeability via regulation of tight junction proteins, and it participates in regulating spermatogenesis, synthesis of steroids and production of the extracellular matrix in testicular tissue. Therefore, it is hypothesized that TGF-ß3 plays important roles in electromagnetic pulse (EMP)-induced changes in BTB permeability. In the present study, we carried out whole-body irradiation on mice using EMP of different intensities. No obvious pathological changes or significant increase in apoptosis was detected in testicular tissues after exposure to 100 and 200 pulses of intensity 200kV/m; however, with 400 pulses we observed the degeneration and shrinkage of testicular tissues along with a significant increase in apoptotic rate. Moreover, in the 100- and 200-EMP groups, a non-significant increase in TGF-ß3 mRNA and protein expression was observed, whereas in the 400-EMP group a significant increase was observed (P<0.05). These results indicate that increase in the apoptotic rate of testicular tissues and increase in TGF-ß3 expression may be one of the mechanisms for EMP-induced increase in BTB permeability in mice.

Nuclear export factor 3 is involved in regulating the expression of TGF-ß3 in an mRNA export activity-independent manner in mouse Sertoli cells.

The NXF (nuclear export factor) family members are implicated in the transport of mRNA from the nucleus to the cytoplasm. Recently, some members of the NXF family have been reported to play divergent functional roles, such as post-transcriptional regulation, translational control, regulation of mRNA stability and trafficking. However, little is known about the roles of NXF3 in spermatogenesis. In the present study, we found that mouse NXF3, specifically expressed in principal cells in segment II of the caput epididymis, as well as Sertoli cells in the mouse testis, was required to mediate TGF-ß (transforming growth factor ß)-induced down-regulation of Tgfb3/TGF-ß3 mRNA expression and protein secretion in Sertoli cells. In addition, NXF3 was also involved in TGF-ß-induced transcriptional regulation of other genes associated with Sertoli cell maturation and the restructuring of the Sertoli cell BTB (blood-testis barrier), such as Gata1 (GATA-binding protein 1), Wt1 (Wilms's tumour homologue 1), Cldn11 (claudin11) and Cdkn1a (cyclin-dependent kinase inhibitor 1A or p21(Cip1)). The transcriptional regulation of NXF3 was mediated through physical interaction with STRAP (serine/threonine kinase receptor-associated protein), where NXF3 inhibited the complex formation among Smad7, STRAP and activated type I TGF-ß receptor. Taken together, our data provide mechanistic insights into the roles of NXF3 in TGF-ß-mediated expression of Tgfb3 and other genes. NXF3 may be implicated in Sertoli cell maturation and the extensive restructuring of the Sertoli cell BTB.

Gene expressional changes in prostate fibroblasts from cancerous tissue.

Prostate cancer is the most common type of cancer in men. It is assumed that the tumor microenvironment of the prostate contributes to invasion and metastasis. Stroma-epithelial crosstalk has shown to change with progression of prostate cancer, and thereby the stromal compartment might be an attractive target in diagnostic and therapeutic approaches to prostate cancer. The purpose of this project was to study the reciprocal influence between fibroblasts and cancer cells in prostate cancer. Prostate fibroblast primary cultures from areas with cancer and hyperplasia were cocultivated with cells of the PC-3 lineage. Gene expression profiles of both cell types were studied to reveal possible associations to cancer invasion and metastasis. There were 383 differentially expressed genes between fibroblasts from cancerous areas and fibroblasts from areas with hyperplasia before cocultivation with PC-3 cells. Several of the differentially expressed gene classes are associated with cancer development and metastasis. After cocultivation, there were 26 differentially expressed genes between cancerous and hyperplastic fibroblasts. There were only three differentially expressed genes between PC-3 cells that had been cocultivated with cancerous fibroblasts and PC-3 cells that had been cocultivated with hyperplastic fibroblasts. The fibroblasts from cancer areas showed a different expression pattern from the characteristics reported as reactive stroma in previous studies. We found tenascin C to be downregulated, which is contrary to previous findings. TGF-ß3 and TGF-ßR3 were also downregulated, which has been associated with disturbance of TGF-ß signaling during prostate cancer progression. Cocultivation with PC-3 cells seems to make the cancerous and hyperplastic fibroblasts more alike each other, as the number of differentially expressed genes decreases. It is desirable to find out if the reduction in differential gene expression is attributable to that hyperplastic fibroblasts become more alike the cancerous fibroblasts or vice versa. Also, we think that the lower expression levels of c-Jun and c-Fos in cancerous fibroblasts without coculture may cause loss of normal fibroblast differentiation, proliferation and inflammatory response, and hence, favor the proliferation and invasion of cancer cells.

TRIM28 prevents autoinflammatory T cell development in vivo.

TRIM28 is a component of heterochromatin complexes whose function in the immune system is unknown. By studying mice with conditional T cell-specific deletion of TRIM28 (CKO mice), we found that TRIM28 was phosphorylated after stimulation via the T cell antigen receptor (TCR) and was involved in the global regulation of CD4(+) T cells. The CKO mice had a spontaneous autoimmune phenotype that was due in part to early lymphopenia associated with a defect in the production of interleukin 2 (IL-2) as well as incomplete cell-cycle progression of their T cells. In addition, CKO T cells showed derepression of the cytokine TGF-ß3, which resulted in an altered cytokine balance; this caused the accumulation of autoreactive cells of the T(H)17 subset of helper T cells and of Foxp3(+) T cells. Notably, CKO Foxp3(+) T cells were unable to prevent the autoimmune phenotype in vivo. Our results show critical roles for TRIM28 in both T cell activation and T cell tolerance.