S1PR2 inhibitors potently reverse 5-FU resistance by downregulating DPD expression in colorectal cancer.

In this study, S1PR2 was reckoned as a brand-new GPCR target for designing inhibitors to reverse 5-FU resistance. Herein a series of pyrrolidine pyrazoles as the S1PR2 inhibitors were designed, synthesized and evaluated for their activities of anti-FU-resistance. Among them, the most promising compound JTE-013, exhibited excellent inhibition on DPD expression and potent anti-FU-resistance activity in various human cancer cell lines, along with the in vivo HCT116DPD cells xenograft model, in which the inhibition rate of 5-FU was greatly increased from 13.01%-75.87%. The underlying mechanism was uncovered that JTE-013 demonstrated an anti-FU-resistance activity by blocking S1PR2 internalization to the endoplasmic reticulum (ER), which inhibited the degradation of 5-FU into α-fluoro-ß-alanine (FBAL) by downregulating tumoral DPD expression. Overall, JTE-013 could serve as the lead compound for the discovery of new anti-FU-resistance drugs. SIGNIFICANCE: This study provides novel insights that S1PR2 inhibitors could sensitize 5-FU therapy in colorectal cancer.

Interleukin-1ß induces hyaluronan and CD44-dependent cell protrusions that facilitate fibroblast-monocyte binding.

Persistent inflammation is a well-known determinant of progressive tissue fibrosis; however, the mechanisms underlying this process remain unclear. There is growing evidence indicating a role of the cytokine IL-1ß in profibrotic responses. We previously demonstrated that fibroblasts stimulated with IL-1ß increased their generation of the polysaccharide hyaluronan (HA) and increased their expression of the HA synthase enzyme (HAS-2). The aim of this study was to determine the significance of IL-1ß-induced changes in HA and HAS-2 generation. In this study, we found that stimulation of fibroblasts with IL-1ß results in the relocalization of HA associated with the cell to the outer cell membrane, where it forms HAS2- and CD44-dependent cell membrane protrusions. CD44 is concentrated within the membrane protrusions, where it co-localizes with the intracellular adhesion molecule 1. Furthermore, we have identified that these cell protrusions enhance IL-1ß-dependent fibroblast-monocyte binding through MAPK/ERK signaling. Although previous data have indicated the importance of the HA-binding protein TSG-6 in maintaining the transforming growth factor ß1-dependent HA coat, TSG-6 was not essential for the formation of the IL-1ß-dependent HA protrusions, thus identifying it as a key difference between IL-1ß- and transforming growth factor ß1-dependent HA matrices. In summary, these data suggest that IL-1ß-dependent HA generation plays a role in fibroblast immune activation, leading to sequestration of monocytes within inflamed tissue and providing a possible mechanism for perpetual inflammation.

Hyaluronan facilitates transforming growth factor-ß1-dependent proliferation via CD44 and epidermal growth factor receptor interaction.

Fibroblast proliferation is an early feature of progressive tissue fibrosis and is largely regulated by the cytokine transforming growth factor-ß1 (TGF-ß1). In the oral mucosa, fibroblasts have a unique phenotype and demonstrate healing with no fibrosis/scarring. Our previous studies show that whereas dermal fibroblasts proliferate in response to TGF-ß1, oral fibroblasts have an antiproliferative response to this cytokine. Hyaluronan (HA) was directly linked to this TGF-ß1-dependent response. The aim of this study was to understand the underlying mechanism through which HA regulates TGF-ß-dependent responses. Using patient-matched oral and dermal fibroblasts, we show that TGF-ß1-dependent proliferation is mediated through the HA receptor CD44, whereas the TGF-ß1-mediated antiproliferative response is CD44-independent. Furthermore, overexpression of HAS2 (HA synthase-2) in oral cells modifies their response, and they subsequently demonstrate a proliferative, CD44-dependent response to TGF-ß1. We also show that epidermal growth factor (EGF) and its receptor (EGFR) are essential for TGF-ß1/HA/CD44-dependent proliferation. Increased HA levels promote EGFR and CD44 coupling, potentiating signal transduction through the MAPK/ERK pathway. Thus, in a HA-rich environment, late ERK1/2 activation results from EGFR/CD44 coupling and leads to a proliferative response to TGF-ß1. In comparison, in a non-HA-rich environment, only early ERK1/2 activation occurs, and this is associated with an antiproliferative response to TGF-ß1. In summary, HA facilitates TGF-ß1-dependent fibroblast proliferation through promoting interaction between CD44 and EGFR, which then promotes specific MAPK/ERK activation, inducing cellular proliferation.

Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.

Bone morphogenetic protein-7 (BMP-7) improves outcome in animal models of fibrotic renal disease by opposing transforming growth factor beta1 (TGF-beta)-dependent fibrosis. However, the underlying mechanisms remain obscure. Here, we studied the effect of BMP-7 on response to TGF-beta in the proximal tubular cell line HK-2 (PTC). BMP-7 specifically limited Smad3 but not Smad2 signaling. BMP-7 did not inhibit Smad3 phosphorylation or nuclear accumulation, nor did BMP-7 alter phosphorylated Smad3 dephosphorylation or degradation. However, BMP-7 treatment reduced Smad3 DNA binding to a consensus Smad binding element probe, and chromatin immunoprecipitation showed reduced Smad3 binding to the plasminogen activator inhibitor-1 promoter in PTCs treated with BMP-7 and TGF-beta compared with TGF-beta alone. Degradation of the transcriptional repressor SnoN has recently been shown to be necessary for Smad3 (but not Smad2) signaling. SnoN expression was transiently lost in PTCs after TGF-beta stimulation, but BMP-7 prevented this. Furthermore, BMP-7 had no effect on Smad3 signaling after siRNA-mediated SnoN knockdown, whereas prevention of SnoN degradation with the proteasome inhibitor MG132 reproduced the inhibitory action of BMP-7 on Smad3 signaling. We conclude that BMP-7 prevents TGF-beta-mediated loss of the transcriptional repressor SnoN and hence specifically limits Smad3 DNA binding, altering the balance of transcriptional responses to TGF-beta in PTCs. These results provide an important mechanistic insight into a key regulator of TGF-beta signaling.

Loss of MicroRNA-192 promotes fibrogenesis in diabetic nephropathy.

The role of microRNAs (miRs), which are endogenous RNA oligonucleotides that regulate gene expression, in diabetic nephropathy is unknown. Here, we performed expression profiling of cultured proximal tubular cells (PTCs) under high-glucose and control conditions. We identified expression of 103 of 328 microRNAs but did not observe glucose-induced changes in expression. Next, we performed miR expression profiling in pooled RNA from formalin-fixed, paraffin-embedded tissue from renal biopsies. We studied three groups of patients with established diabetic nephropathy and detected 103 of 365 miRs. Two miRs differed by more than two-fold between progressors and nonprogressors, and 12 miRs differed between late presenters and other biopsies. We noted the greatest change in miR-192 expression, which was significantly lower in late presenters. Furthermore, in individual biopsies, low expression of miR-192 correlated with tubulointerstitial fibrosis and low estimated GFR. In vitro, treatment of PTCs with TGF-beta1 decreased miR-192 expression. Overexpression of miR-192 suppressed expression of the E-Box repressors ZEB1 and ZEB2, thereby opposing TGF-beta-mediated downregulation of E-cadherin. In summary, loss of miR-192 expression associates with increased fibrosis and decreased estimated GFR in diabetic nephropathy in vivo, perhaps by enhancing TGF-beta-mediated downregulation of E-cadherin in PTCs.

SphK2 confers 5-fluorouracil resistance to colorectal cancer via upregulating H3K56ac-mediated DPD expression.

Aberrant sphingolipid metabolism has been implicated in chemoresistance, but the underlying mechanisms are still poorly understood. Herein we revealed a previously unrecognized mechanism of 5-fluorouracil (5-FU) resistance contributed by high SphK2-upregulated dihydropyrimidine dehydrogenase (DPD) in colorectal cancer (CRC), which is evidenced from human CRC specimens, animal models, and cancer cell lines. TMA samples from randomly selected 60 CRC specimens firstly identified the clinical correlation between high SphK2 and increased DPD (p < 0.001). Then the regulatory mechanism was explored in CRC models of villin-SphK2 Tg mice, SphK2-/-mice, and human CRC cells xenografted nude mice. Assays of ChIP-Seq and luciferase reporter gene demonstrated that high SphK2 upregulated DPD through promoting the HDAC1-mediated H3K56ac, leading to the degradation of intracellular 5-FU into inactive α-fluoro-ß-alanine (FBAL). Lastly, inhibition of SphK2 by SLR080811 exhibited excellent inhibition on DPD expression and potently reversed 5-FU resistance in colorectal tumors of villin-SphK2 Tg mice. Overall, this study manifests that SphK2high conferred 5-FU resistance through upregulating tumoral DPD, which highlights the strategies of blocking SphK2 to overcome 5-FU resistance in CRC.

Interleukin-1 beta regulates proximal tubular cell transforming growth factor beta-1 signalling.

BACKGROUND: Increased transforming growth factor beta-1 (TGF beta) expression in the kidney is central not only to the pathogenesis of tubulointerstitial fibrosis but also in repair following acute injury. Recent work suggests that pro-inflammatory cytokines may determine epithelial cell responses to TGF beta in the contexts of acute injury and chronic wounding. METHODS: In this study, we examined the effects of interleukin-1 beta (IL-1) on proximal tubular cell (PTC) response to TGF beta. RESULTS: IL-1 induced the rapid activation of NF-kappaB in PTC. This was associated with inhibition of Smad2 and Smad3 signalling. NF-kappaB activation by IL-1 was transient, with a change from p65/p50 heterodimer to p50/p50 homodimer formation by 24 h and a switch to enhanced Smad signalling response to TGF beta. This was associated with IL-6 generation and prevented by IL-6 receptor blockade. CONCLUSION: In summary, IL-1 has a biphasic effect on PTC TGF beta signalling, with early NF-kappaB-mediated inhibition and delayed sensitization via an autocrine IL-6 loop. These results provide mechanistic insight into how acute and chronic inflammation help define epithelial cell response to TGF beta, and hence how TGF beta can have apparently contradictory roles, being involved in controlled healing following acute injury on one hand, yet the principal promoter of scarring in chronic disease on the other.

Structural modification of oridonin via DAST induced rearrangement.

A simple and efficient protocol was developed for the syntheses of oridonin analogues, i.e. 6,20-epoxy ent-kaurane diterpenoid analogues from oridonin via diethylaminosulfur trifluoride (DAST) promoted rearrangement, most of which exhibited superior anticancer activities compared with their precursor.

Connective tissue growth factor is associated with the early renal hypertrophy in uninephrectomized diabetic rats.

BACKGROUND: Renal hypertrophy has been regarded as the early feature of diabetic nephropathy (DN), which may eventually lead to proteinuria and renal fibrosis. However, the exact mechanism of renal hypertrophy is still unclear. The aim of this study was to investigate the possible association of connective tissue growth factor (CTGF) with renal hypertrophy in uninephrectomized diabetic rats. METHODS: Seventy-two Sprague-Dawley (SD) rats were randomly divided into two groups: control group (group C, n = 32) and diabetic nephropathy (group DN, n = 40). Each group was re-divided into 4 subgroups according to the experimental period. The rats were sacrificed at 1, 2, 4, and 8 weeks respectively after induction of diabetes. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) after rats had received uninephrectomy. Blood glucose (BG), body weight (BW), 24-h urinary albumin excretion (24hUalb), kidney weight (KW), KW/BW, glomerular tuft area (AG), glomerular tuft volume (VG), proximal tubular area (AT) at each time point, the width of glomerular basement membrane (GBM) and tubular basement membrane (TBM) at week 8 were measured when the rats were sacrificed. Renal expression of CTGF and p27kip1 were detected by immunohistochemical staining. The relationship between CTGF expression and increasing of VG and AT was analyzed. RESULTS: There was a significant increase of 24hUalb, KW, and KW/BW from week 1 onward in diabetic rats compared to those in group C (P < 0.05, respectively), diabetic rats also had a significant increase of AG, VG, and AT from week 1 onward. It was also shown that diabetic rats had a thickening of GBM [(245.7 +/- 103.0) nm vs (121.8 +/- 19.1) nm, P < 0.01] and TBM [(767.7 +/- 331.1) nm vs (293.0 +/- 110.5) nm, P < 0.01] at week 8. There was a weak expression for CTGF and p27kip1 in normal glomeruli and tubuli, while a significant increasing expression of CTGF and p27kip1 was found in glomeruli and tubuli in diabetic kidney from week 1 onward (P < 0.05, respectively), and the extent of CTGF expression was positively correlated with AG (r = 0.92, P < 0.05), VG (r = 0.86, P < 0.05), AT (r = 0.94, P < 0.01) and positively correlated with the expression of p27kip1 (r = 0.96, P < 0.01). CONCLUSION: The expression of CTGF increases in diabetic rat kidney at the early stage, which might be an important mediator of renal hypertrophy through arresting cell cycling.

[Treatment of fresh Monteggia fractures of Bado type I and II in children by closed reduction and ulna intramedullary nail fixation].

OBJECTIVE: To investigate the effects of closed reduction and ulna intramedullary nail fixation for the treatment of fresh Monteggia fractures of Bado type I and II in children. METHODS: Twenty-three children patients with Monteggia fracture during July 2010 to September 2013 were treated by closed reduction and ulna intramedullary nail fixaion including 18 boys and 5 girls with an average age of 9.3 years old ranging from 6 to 13 years old. Among them,15 cases were Bado type I and 8 cases were Bado type II. There were 9 cases with radial nerve injury. The operation time,the recovery of nerve injury, the fracture healing and the function of elbow were observed and recorded. RESULTS: All patients were followed up for 6 to 24 months (12 months on average). All patients were obtained bone healing. According to Anderson standard, at the final follow-up, 20 cases got excellent result, 2 cases got good result, and one case got fair result. CONCLUSION: Treatment of the fresh Monteggia fractures in children by closed reduction and ulna intramedullary nail fixation has advantages of simple operation, less trauma and good results.

[Closed reduction and three Kirschner pin fixation at the radial side for the treatment of supracondylar fracture in children with Gartland type II and type III].

OBJECTIVE: To investigate the characteristics of supracondylar fracture of humerus in children and to explore the effect of closed reduction and internal fixation at radial side on the fracture. METHODS: The children with fractures of Gartland type II and type III in our hospital from June 2010 to June 2013 were reviewed. There were 28 males and 7 females, ranging in age from 1 year and 1 month to 2 years and 6 months, with an average of 2 years and 1 month. According to Gartland classification, 19 cases were type II, 16 cases were type III. There were 3 patients with radial nerve injuries, and 5 patients with anterior interosseous nerve injuries. There were no vascular injuries. All the patients were treated with closed reduction and three Kirschner fixation at the radial side, followed by the plaster external fixation with elbowed flexion at 90 °. The X-ray examination was performed at the second day after operation. The joint function exercise began about at 2 to 3 weeks after operation when the plaster fixation was removed, and opportune time for removal of Kirschners depends on the situation of fracture healing. The operation time, nerve recovery, and the elbow joint function were observed. RESULTS: All the children were followed up, and all the fractures had bony union. According to Flynn score system at the final follow-up, 28 patients got an excellent result, 4 good, 1 poor and 2 bad. CONCLUSION: Three Kirschner fixation at the radial side for the treatment of supracondylar fracture of humerus has advantages of minimally invasive, shorter time of hospitalization, simple removal of the internal fixation and reliable therapeutic effects.

Effect of irbesartan on angiotensin II-induced hypertrophy of human proximal tubular cells.

BACKGROUND: Intrarenal activation of the renin angiotensin system (RAS) plays an important role in mediating renal fibrosis. Both angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II (AngII) receptor antagonists have been shown to exert a protective role against diabetic and non-diabetic nephropathy. However, the exact mechanism of how blocking local RAS prevents renal fibrosis is unclear. The present study was to investigate the influence of a new AngII receptor antagonist, irbesartan (Irb), on AngII-induced hypertrophy in human proximal tubular cell line (HK-2). METHODS: The cell line, HK-2, was grown in Dulbeccos's Modified Eagle's Medium containing 10% heat-inactivated fetal calf serum. After rested in serum-free medium for 24 hours, the effects of Irb on AngII (10(-7) mol/L)-induced [(3)H]-leucine incorporation, total protein content (measured by the Coomassie brilliant blue G250 method), and change in cell size (determined by scanning electron microscopy) were observed. The influence of Irb on the cell cycle was analyzed by fluorescence activated cell sorter (FACS) flow cytometry. RESULTS: AngII induced cell hypertrophy in a time and dose dependent manner. Stimulation of cells with AngII for 48 hours resulted in a increase in [(3)H]-leucine incorporation [0 hour: (5584 +/- 1016) cpm/10(5) cells vs 48 hours: (10741 +/- 802) cpm/10(5) cells, P < 0.05], which was significantly attenuated by treatment with Irb. AngII significantly increased the total protein content in HK-2 cells [control: (0.169 +/- 0.011) mg/10(5) cells vs AngII group: (0.202 +/- 0.010) mg/10(5) cells, P < 0.05], which was also markedly inhibited by cotreatment with Irb (P < 0.01). Scanning electron microscopy showed that AngII induced an increase in average physical cell size, which was significantly inhibited by Irb [control: (11.92 +/- 1.62) microm; AngII group: (20.63 +/- 3.83) micro m; AngII + Irb group: (13.59 +/- 3.15) micro m; P < 0.01 vs control, respectively]. Furthermore, flow cytometry revealed that AngII arrested cells in the G(0)-G(1) phase, which was significantly reversed by treatment with Irb [G(0)-G(1) cells in AngII group: (76.09 +/- 1.82)%, in AngII + Irb group: (67.00 +/- 2.52)%, P < 0.05]. CONCLUSION: Irb can inhibit AngII-induced hypertrophy in HK-2 cells.

[Influence of irbesartan on renal hypertrophy and thickening of glomerular basement-membrane in streptozotocin-induced diabetic rats].

OBJECTIVE: To investigate the influence of angiotensin II receptor antagonist irbesartan (Irb) on renal hypertrophy and thickening of glomerular basement membrane (GBM) in streptozotocin (STZ) induced diabetic rats. METHODS: Sprague-Dalwley (SD) rats were randomly divided into three groups: normal control (group N, n = 7), diabetic nephropathy (group DN, n = 6) and diabetic nephropathy treated with Irb (group DNI, n = 7). Diabetes was induced by injection of STZ intraperitoneally after rats had received uninephroectomy. Blood glucose (BG), body weight (BW), urinary albumin excretion (Ualb), 24-hour proteinuria (24hUpro) were observed in the rats at week 4, 8, 12 respectively. Creatinine clearance (Ccr), kidney weight (KW), profile of kidney hypertrophy (KW/BW), renal tissue protein contents (RTP), glomerular area (A(G)), glomerular volume (V(G)), and width of GBM were determined at week 12 when the rats were sacrificed. Renal expression of connective tissue growth factor (CTGF) and transforming growth factor-beta(1) (TGF-beta(1)) were determined by immunohistochemistry. RESULTS: There is no significant difference of BG between group DN and group DNI (P > 0.05). Irb treatment significantly prevented the increase of Ualb excretion and 24hUpro and the deterioration of Ccr in diabetic rats (P < 0.05, P < 0.01 respectively). Furthermore, Irb markedly inhibited the increasing of KW, KW/BW, RTP, AG and VG shown in diabetic rats (P < 0.05, P < 0.01 respectively). More interestingly, we firstly demonstrated that Irb significantly prevented the thickening of GBM [N group: (127.50 +/- 22.14) nm, DN group: (280.38 +/- 52.77) nm, DNI group: (144.07 +/- 24.85) nm] and immunostaining for CTGF (P < 0.05 respectively). In addition, the extent of CTGF expression is positively correlated with the glomerular immunostaining for TGF-beta(1) and size of V(G) (P < 0.01). CONCLUSION: This is the first data to demonstrate that Irb exerts early renal protective role in diabetic nephropathy, possibly through inhibition of renal hypertrophy and renal expression of CTGF.

Identification and analysis of the human hyaluronan synthase 1 gene promoter reveals Smad3- and Sp3-mediated transcriptional induction.

The ubiquitous mammalian extracellular matrix glycosaminoglycan hyaluronan (HA) plays a pivotal role in the regulation of cell phenotype in fibrosis and scarring. Transforming growth factor-beta 1 (TGF-ß1) and interleukin-1 beta (IL-1ß) up-regulate hyaluronan synthase (HAS) 1 and HAS2 in dermal fibroblasts and renal proximal tubular epithelial cells, and subsequent HA synthesis regulates cell phenotype. In the present study, we investigated the mechanism of HAS1 transcriptional up-regulation in response to these cytokines. We used 5'-rapid amplification of cDNA ends analysis to identify the 5' end of HAS1 transcripts, resulting in an increase of 26 nucleotides to the HAS1 exon 1 sequence of reference sequence NM_001523. Constitutive luciferase activity of upstream DNA sequences was shown in luciferase reporter assays, but our reporter vector signals were refractory to the addition of TGF-ß1 and IL-1ß. Using siRNAs to knockdown transcription factor mRNAs, we showed that TGF-ß1 up-regulation of HAS1 transcription was mediated via Smad3 but not Smad2, while HAS1 induction by IL-1ï€ ß was Sp3, not Sp1, dependent.

[Design and application of inflatable pressure applicator on the treatment of femoral shaft fractures: case control study].

OBJECTIVE: To compare the influence on the postoperative wound healing between inflatable pressure applicator and traditional pressure dressing. METHODS: From May 2009 to February 2010, 50 patients with closed femoral shaft fractures were randomly divided into group A and group B, with 25 patients in each group. There were 13 males and 12 females in group A, ranging in age from 38 to 60 years, with an average of (55.1 +/- 9.5) years; of them, road accident was in 18 cases, fall from heigh was in 6 cases, accidental falling injury was in 1 case. There were 15 males and 10 females in group B, ranging in age from 40 to 65 years, with an average of (56.5 +/- 9.2) years; of them, road accident was in 13 cases, fall from heigh was in 6 cases, accidental falling injury was in 6 cases. There was no significant difference between two groups in clinical data. The postoperative wounds of group A were binded with dressing of inflatable pressure applicator; and of group B with traditional pressure dressing. Volume of drainage at 12 h after operation, saturation of blood oxygen at 12, 24 h after operation, satisfactory rate of patients were compared between two groups. RESULTS: Postoperative volume of drainage in group A was lower than that of group B, respectively was (77.5 +/- 4.6), (94.3 +/- 3.8) ml. Saturation of blood oxygen at 12, 24 h after operation in A group was respectively (98.3 +/- 1.1)%, (98.9 +/- 0.8)%, and in group B was respectively (96.5 +/- 0.4)%, ( 97.0 +/- 0.3)%; there was significant difference between two groups at the same time. Satisfactory rate of patients in A group was better than that of group B. CONCLUSION: Inflatable pressure applicator can obviously pressurize and stop bleeding for postoperative wounds, but no affect on peripheral blood supply and can improve discomfort of patients.

[Microsurgical treatment of intramedullary tumor in the superior cervical spinal cord].

OBJECTIVE: To evaluate the curative effect of microsurgery for intramedullary tumor in the superior cervical spinal cord. METHODS: The clinical manifestations, microsurgical methods and results were reviewed retrospectively in 12 patients with intramedullary tumors in the superior cervical spinal cord. RESULTS: No death occurred in these cases after the operations. The intramedullary tumors were totally resected in 10 patients including 8 with ependymomas and 2 with astrocytomas, and subtotally in 2 patients with astrocytomas. The spinal functions of patients, graded by McCormick scale system 3 months after the operations, were improved in 8 cases and remained unchanged in 4 cases. Nine patients were followed up for 1-3 years after the operations, and no tumor recurrence was found in 8 cases with total tumor resection. CONCLUSION: Radical microneurosurgery is currently the best choice for the treatment of intradullary tumor in the superior cervical spinal cord.

Polarity of response to transforming growth factor-beta1 in proximal tubular epithelial cells is regulated by beta-catenin.

Transforming growth factor-beta1 (TGF-beta1)-mediated loss of proximal tubular epithelial cell-cell interaction is regulated in a polarized fashion. The aim of this study was to further explore the polarity of the TGF-beta1 response and to determine the significance of R-Smad-beta-catenin association previously demonstrated to accompany adherens junction disassembly. Smad3 signaling response to TGF-beta1 was assessed by activity of the Smad3-responsive reporter gene construct (SBE)(4)-Lux and by immunoblotting for phospho-Smad proteins. Similar results were obtained with both methods. Apical application of TGF-beta1 led to increased Smad3 signaling compared with basolateral stimulation. Association of Smad proteins with beta-catenin was greater following basolateral TGFbeta-1 stimulation, as was the expression of cytoplasmic Triton-soluble beta-catenin. Inhibition of beta-catenin expression by small interfering RNA augmented Smad3 signaling. Lithium chloride, a GSK-3 inhibitor, increased expression of beta-catenin and attenuated TGF-beta1-dependent Smad3 signaling. Lithium chloride did not influence degradation of Smad3 but resulted in decreased nuclear translocation. Smad2 activation as assessed by Western blot analysis and activity of the Smad2-responsive reporter constructs ARE/MF1 was also greater following apical as compared with basolateral TGFbeta-1 stimulation, suggesting that this is a generally applicable mechanism for the regulation of TGF-beta1-dependent R-Smads. Caco-2 cells are a colonic carcinoma cell line, with known resistance to the anti-proliferative effects of TGF-beta1 and increased expression of beta-catenin. We used this cell line to address the general applicability of our observations. Inhibition of beta-catenin in this cell line by small interfering RNA resulted in increased TGF-beta1-dependent Smad3 phosphorylation and restoration of TGF-beta1 anti-proliferative effects.

Sp1 and Sp3 mediate constitutive transcription of the human hyaluronan synthase 2 gene.

The linear glycosaminoglycan hyaluronan (HA) is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2, and -3 and performs multiple functions as part of the vertebrate extracellular matrix. Up-regulation of HA synthesis in the renal corticointerstitium, and the resultant extracellular matrix expansion, is a common feature of renal fibrosis. However, the regulation of expression of these HAS isoforms at transcriptional and translational levels is poorly understood. We have recently described the genomic structures of the human HAS genes, thereby identifying putative promoter regions for each isoform. Further analysis of the HAS2 gene identified the transcription initiation site and showed that region F3, comprising the proximal 121 bp of promoter sequence, mediated full constitutive transcription. In the present study, we have analyzed this region in the human renal proximal tubular epithelial cell line HK-2. Electrophoretic mobility shift and promoter assay data demonstrated that transcription factors Sp1 and Sp3 bound to three sites immediately upstream of the HAS2 transcription initiation site and that mutation of the consensus recognition sequences within these sites ablated their transcriptional response. Furthermore, subsequent knockdown of Sp1 or Sp3 using small interfering RNAs decreased constitutive HAS2 mRNA synthesis. In contrast, significant binding of HK-2 nuclear proteins by putative upstream NF-Y, CCAAT, and NF-kappaB recognition sites was not observed. The identification of Sp1 and Sp3 as principal mediators of HAS2 constitutive transcription augments recent findings identifying upstream promoter elements and provides further insights into the mechanism of HAS2 transcriptional activation.

Mechanisms of irbesartan in prevention of renal lesion in streptozotocin-induced diabetic rats.

AIM: To investigate the mechanisms of angiotensin II receptor antagonist irbesartan (Irb) in prevention of renal lesion in streptozotocin (STZ)-induced diabetic rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into three groups: normal control (group N), diabetic nephropathy (group DN), and diabetic nephropathy treated with Irb (group DNI). Diabetes was induced by injection of STZ ip after rats had received uninephrectomy. Blood glucose (BG), body weight (BW), urinary albumin excretion (Ualb), and 24-h proteinuria (24hUpro) were observed in the rats at week 4, 8, and 12, respectively. Creatinine clearance (Ccr), the kidney weight (KW), profile of kidney hypertrophy (KW/BW), renal tissue protein contents (RTP), glomerular area (AG), glomerular volume (VG), and width of glomerular basement membrane (GBM) were determined after the rats were sacrificed at week 12. Renal expression of connective tissue growth factor (CTGF) and transforming growth factor-beta1 (TGF-beta1) were determined by immunohistochemistry. RESULTS: There was no significant difference in BG between group DN and DNI (P >0.05). Irb prevented the increasing of Ualb excretion, 24 hUpro, and Ccr in diabetic rats ( P < 0.01). Furthermore, Irb markedly inhibited the increasing of KW, KW/BW, RTP, AG, and VG shown in diabetic rats (P <0.05, P <0.01, respectively). Irb prevented the thickening of GBM and immunostaining of CTGF (P <0.01). The extent of CTGF expression was positively correlated with the glomerular immunostaining for TGF-beta1 and size of VG (P <0.01). CONCLUSION: Irb exerts an early renal protective role to diabetic nephropathy, possibly through inhibition of renal hypertrophy and expression of CTGF.