LPCAT4 Knockdown Alters Barrier Integrity and Cellular Bioenergetics in Human Urothelium.

Urothelium is a transitional, stratified epithelium that lines the lower urinary tract, providing a tight barrier to urine whilst retaining the capacity to stretch and rapidly resolve damage. The role of glycerophospholipids in urothelial barrier function is largely unknown, despite their importance in membrane structural integrity, protein complex assembly, and the master regulatory role of PPARγ in urothelial differentiation. We performed lipidomic and transcriptomic characterisation of urothelial differentiation, revealing a metabolic switch signature from fatty acid synthesis to lipid remodelling, including 5-fold upregulation of LPCAT4. LPCAT4 knockdown urothelial cultures exhibited an impaired proliferation rate but developed elevated trans-epithelial electrical resistances upon differentiation, associated with a reduced and delayed capacity to restitute barrier function after wounding. Specific reduction in 18:1 PC fatty acyl chains upon knockdown was consistent with LPCAT4 specificity, but was unlikely to elicit broad barrier function changes. However, transcriptomic analysis of LPCAT4 knockdown supported an LPC-induced reduction in DAG availability, predicted to limit PKC activity, and TSPO abundance, predicted to limit endogenous ATP. These phenotypes were confirmed by PKC and TSPO inhibition. Together, these data suggest an integral role for lipid mediators in urothelial barrier function and highlight the strength of combined lipidomic and transcriptomic analyses for characterising tissue homeostasis.

Targeting Deficiencies in the TLR5 Mediated Vaginal Response to Treat Female Recurrent Urinary Tract Infection.

The identification of the host defence peptides as target effectors in the innate defence of the uro-genital tract creates new translational possibilities for immunomodulatory therapies, specifically vaginal therapies to treat women suffering from rUTI, particularly those carrying the TLR5_C1174T SNP. Urinary tract infections (UTIs) are a microbial disease reported worldwide. Women are particularly susceptible with many suffering debilitating recurrent (r) infections. Treatment is by antibiotics, but such therapy is linked to antibiotic resistance and re-infection. This study explored the innate protective mechanisms of the urogenital tract with the aim of boosting such defences therapeutically. Modelling UTIs in vitro, human vaginal and bladder epithelial cells were challenged with uropathogenic Escherichia coli (CFT073) and microbial PAMPs including flagellin, LPS and peptidoglycan. Flagellin functioning via the TLR5/NFκB pathway was identified as the key UPEC virulence factor causing a significant increase (P < 0.05) in the production of the host-defence peptide (HDP), BD2. BD2-depleted urine samples from bladder infected mice supported increased UPEC growth, strengthening the significance of the HDPs in protecting the urogenital tissues from infection. Clinically, vaginal-douche BD2 concentrations were reduced (p < 0.05) in women suffering rUTIs, compared to age-matched healthy controls with concentrations further decreased (p < 0.05) in a TLR5392Stop SNP rUTI subgroup. Topical vaginal estrogen treatment increased (p < 0.001) BD2 concentrations in all women, including those carrying the SNP. These data identify therapeutic and antibiotic sparing roles for vaginal immunomodulatory agents that specifically target HDP induction, facilitate bacterial killing and disrupt the UPEC infection cycle.

The human urothelial tight junction: claudin 3 and the ZO-1α+ switch.

OBJECTIVE: Tight junctions are multicomponent structures, with claudin proteins defining paracellular permeability. Claudin 3 is a candidate for the exceptional "tightness" of human urothelium, being localised to the terminal tight junction (TJ) of superficial cells. Our aim was to determine whether claudin 3 plays an instigating and/or a functional role in the urothelial TJ. MATERIALS AND METHODS: Normal human urothelial (NHU) cells maintained as non-immortalised cell lines were retrovirally-transduced to over-express or silence claudin 3 expression. Stable sublines induced to stratify or differentiate were assessed for TJ formation by immunocytochemistry and transepithelial electrical resistance (TER). Expression of claudin 3, ZO-1 and ZO-1α+ was examined in native urothelium by immunohistochemistry. RESULTS: Claudin 3 expression was associated with differentiation and development of a tight barrier and along with ZO-1 and ZO-1α+ was localised to the apical tight junction in native urothelium. Knockdown of claudin 3 inhibited formation of a tight barrier in three independent cell lines, however, overexpression of claudin 3 was not sufficient to induce tight barrier development in the absence of differentiation. A differentiation-dependent induction of the ZO-1α+ isoform was found to coincide with barrier formation. Whereas claudin 3 overexpression did not induce the switch to co-expression of ZO-1α-/ZO-1α+, claudin 3 knockdown decreased localisation of ZO-1 to the TJ and resulted in compromised barrier function. CONCLUSIONS: Urothelial cytodifferentiation is accompanied by induction of claudin 3 which is essential for the development of a terminal TJ. A coordinated switch to the ZO-1α+ isotype was also observed and for the first time may indicate that ZO-1α+ is involved in the structural assembly and function of the urothelial terminal TJ.

Differentiation-associated reprogramming of the transforming growth factor ß receptor pathway establishes the circuitry for epithelial autocrine/paracrine repair.

Transforming growth factor (TGF) ß has diverse and sometimes paradoxical effects on cell proliferation and differentiation, presumably reflecting a fundamental but incompletely-understood role in regulating tissue homeostasis. It is generally considered that downstream activity is modulated at the ligand:receptor axis, but microarray analysis of proliferative versus differentiating normal human bladder epithelial cell cultures identified unexpected transcriptional changes in key components of the canonical TGFß R/activin signalling pathway associated with cytodifferentiation. Changes included upregulation of the transcriptional modulator SMAD3 and downregulation of inhibitory modulators SMURF2 and SMAD7. Functional analysis of the signalling pathway revealed that non-differentiated normal human urothelial cells responded in paracrine mode to TGFß by growth inhibition, and that exogenous TGFß inhibited rather than promoted differentiation. By contrast, in differentiated cell cultures, SMAD3 was activated upon scratch-wounding and was involved in promoting tissue repair. Exogenous TGFß enhanced the repair and resulted in hyperplastic scarring, indicating a feedback loop implicit in an autocrine pathway. Thus, the machinery for autocrine activation of the SMAD3-mediated TGFßR pathway is established during urothelial differentiation, but signalling occurs only in response to a trigger, such as wounding. Our study demonstrates that the circuitry of the TGFßR pathway is defined transcriptionally within a tissue-specific differentiation programme. The findings provide evidence for re-evaluating the role of TGFßR signalling in epithelial homeostasis as an autocrine-regulated pathway that suppresses differentiation and promotes tissue repair. This provides a new paradigm to help unravel the apparently diverse and paradoxical effect of TGFß signalling on cell proliferation and differentiation.

Toll-like receptor responses of normal human urothelial cells to bacterial flagellin and lipopolysaccharide.

PURPOSE: We determined toll-like receptor expression in normal human urothelium and functional responses in normal human urothelial cell cultures to bacterial lipopolysaccharide via toll-like receptor-4 and to flagellin via toll-like receptor-5. MATERIALS AND METHODS: Toll-like receptor protein expression was examined immunohistochemically. Toll-like receptor transcript expression was determined in freshly isolated urothelium, and in proliferating and differentiated normal human urothelial cultured cells. Lipopolysaccharide binding was assessed by flow cytometry. Functional responses of proliferating and differentiated normal human urothelial cells to lipopolysaccharide and flagellin were determined by interleukin-6 and 8 secretion, and transcription factor activation. Polymyxin B and siRNA were used to confirm the specificity of toll-like receptor-4 and 5 responses, respectively. Western blot detection of phosphorylated IκB was used to confirm toll-like receptor-4 results. RESULTS: Human urothelium expressed transcripts for toll-like receptor-4 and 5. Although bladder cancer derived T24 cells responded to lipopolysaccharide, there was no lipopolysaccharide binding to normal human urothelial cells and no functional response of proliferative or differentiated normal human urothelial cells even in the presence of exogenous CD14 and MD-2 accessory proteins. In contrast, flagellin evoked a toll-like receptor-5 mediated response in proliferating but not in differentiated normal human urothelial cells, which was abrogated by toll-like receptor-5 specific siRNA. CONCLUSIONS: Results suggest that human urothelium may mediate a host response to uropathogenic Escherichia coli through the detection of flagellin. The absent constitutive toll-like receptor-4 response may reflect an adaptation of urothelium toward sustaining barrier function and limiting inflammation to soluble bacterial products.

Immortalisation of normal human urothelial cells compromises differentiation capacity.

BACKGROUND: The development of urothelial malignancy is not solely a consequence of loss of proliferation constraints but also involves loss of cellular differentiation, defined histopathologically as grade. Although tumour grade is an independent prognostic marker for urothelial carcinoma (UC), the molecular events underpinning the loss of urothelial differentiation are poorly understood. OBJECTIVE: To examine the effect of gene alterations implicated in UC development on the ability of human urothelial cells to undergo molecular differentiation and form a functional urothelial barrier. DESIGN, SETTING, AND PARTICIPANTS: Laboratory study. INTERVENTION: Normal human urothelial (NHU) cell cultures were transduced with recombinant retroviruses to produce stable sublines overexpressing wild-type or oncogenic mutated fibroblast growth factor receptor 3 or human telomerase reverse transcriptase (hTERT). Previously generated NHU sublines carrying dominant-negative CDK4 and p53 mutant genes or immortalised with the human papillomavirus 16 E6 oncoprotein were included. MEASUREMENTS: The activity of introduced transgenes was demonstrated by comparing phenotypes of transgene-expressing and isogenic control NHU cells. Modified and control sublines were compared for changes in generational potential (life span) and capacity to respond to differentiation-inducing signals by transcript expression of uroplakins 2 and 3. The ability to form a barrier epithelium was assessed by measuring the transepithelial electrical resistance. RESULTS AND LIMITATIONS: By contrast to tumour suppressor loss of function or oncogene overactivation, hTERT overexpression alone led to life span extension and immortalisation. The hTERT immortalised cells carried no gross genomic alterations but became progressively insensitive to differentiation signals and lost the ability to form an epithelial barrier. Further characterisation of hTERT cells revealed a downregulation of p16 cyclin-dependent kinase inhibitor expression and loss of responsiveness to peroxisome proliferator-activated receptor γ, providing mechanistic explanations for the subjugation of senescence constraints and the abrogation of differentiation capability, respectively. Although immortalised urothelial cell lines without karyotypic aberrations may be generated, such cell lines are compromised in terms of differentiation and functional capacity. CONCLUSIONS: Overexpression of hTERT promotes development of an immortalised differentiation-insensitive urothelial cell phenotype. Although such cells offer a useful insight into the grade/stage paradigm of UC, they have limited value for investigating normal urothelial cell/tissue biology and physiology.

Organotypic and 3D reconstructed cultures of the human bladder and urinary tract.

Three-dimensional organotypic cultures of human urinary tract tissue have been established as intact and reconstituted tissues, with the latter generated by combining cultured normal human urothelial (NHU) cells with an appropriate stroma. Organoids may be maintained at an air-liquid interface in static culture for periods of up to 20 weeks, with analysis by immunohistology for expression of urothelial differentiation-associated markers providing a qualitative, but objective assessment criterion. Where reconstructed using bladder cancer cell lines, the resultant organoids recapitulate the invasive characteristics of the originating tumour, but the need to use authenticated cell line stocks is emphasised. The organoid approach represents an important tool for investigating urothelial-stromal cell interactions during homeostasis and disease, and for testing bladder tissue engineering and reconstructive strategies. Potential future developments of the technique are discussed and include genetic manipulation of the urothelial cells to generate disease models and incorporation of biomaterial scaffolds to support artificial stroma development.

Effects of PPAR agonists on proliferation and differentiation in human urothelium.

Systemic treatment of rats with peroxisome proliferator-activated receptor (PPAR) agonists (mainly of dual alpha/gamma activity) has indicated that they may invoke non-genotoxic carcinogenesis in the epithelial lining of the urinary tract (urothelium). Although there is evidence in the male rat to support an indirect effect via a crystaluria-induced urothelial damage response, there is other evidence to indicate a direct signalling effect on the urothelium and hence the full implication for using these drugs in man is unclear. Numerous reports have demonstrated that PPARs are expressed within the urothelium of different species, including man, and from an early developmental stage. We have developed methods to maintain normal human urothelial (NHU) cells in culture, where the cells retain PPAR expression and express a highly proliferative phenotype, mediated via autocrine stimulation of the epidermal growth factor (EGF) receptor. We have shown that specific activation of PPARgamma results in a programme of gene expression changes associated with late/terminal cytodifferentiation, including induction of cytokeratins CK13 and CK20, tight junction-associated claudin 3, and uroplakins UPK1a and UPK2, but this is dependent upon inhibition of the signalling cascade downstream of the EGF receptor. This indicates a subtle balance in the regulation of proliferation and differentiation in urothelium, with PPARgamma agonists promoting differentiation. Our data indicate that human urothelium is a target tissue for PPARgamma signalling, but it has yet to be determined whether dual agonists could have a modulatory effect on the proliferation/differentiation balance.

Differentiation potential of urothelium from patients with benign bladder dysfunction.

OBJECTIVE: To develop a novel in vitro approach to test the hypothesis that failure of urothelial differentiation underlies the aetiopathology of interstitial cystitis (IC), where there is evidence of compromised urinary barrier function, as benign dysfunctional bladder disease encompass several poorly understood clinically defined conditions, including IC, idiopathic detrusor overactivity (IDO) and stress urinary incontinence (SUI). MATERIALS AND METHODS: Biopsy-derived urothelial cells from dysfunctional bladder biopsies were propagated as finite cell lines and examined for their capacity to differentiate in vitro, as assessed by the acquisition of a transitional cell morphology, a switch from a cytokeratin (CK)13(lo)/CK14(hi) to a CK13(hi)/CK14(lo) phenotype, expression of claudin 3, 4 and 5 proteins, and induction of uroplakin gene transcription. RESULTS: Two of 12 SUI cell lines showed early senescent changes in culture and were not characterized further; one of seven IC, one of five IDO and a further three SUI cell lines had some evidence of senescence at passage 3. Of the seven IC-derived cell lines, four showed a near normal range of differentiation-associated responses, but the remainder showed little or no response. Most IDO cell lines (four of five) showed a normal differentiation response, but at least three of the 10 SUI cell lines showed some compromise of differentiation potential. CONCLUSION: This study supports the existence of a subset of patients with IC in whom a failure of urothelial cytodifferentiation might contribute to the disease, and provides a novel platform for investigating the cell biology of urothelium from SUI and other benign dysfunctional conditions.

PPARgamma-regulated tight junction development during human urothelial cytodifferentiation.

Urothelial barrier function is maintained by apical membrane plaques and intercellular tight junctions (TJ). Little is known about the composition and regulation of TJ expression in human urothelium. In this study, we have characterised the expression of TJ components in situ and their regulation in an in vitro model of differentiating normal human urothelial (NHU) cells. In normal ureteric urothelium in situ, there was a differentiation-associated profile of claudins 3, 4, 5, 7, ZO1 and occludin proteins. Proliferating NHU cells in vitro expressed predominantly claudin 1 protein and transcripts for claudins 1-5 and 7. Following induction of differentiation by pharmacological activation of PPARgamma and blockade of EGFR, there was de novo expression of claudin 3 mRNA and protein and downregulation of claudin 2 transcription. There was also a massive increase in expression of claudin 4 and 5 proteins which was due to inhibition of proteasomal degradation of claudin 4 and consequential stabilisation of the claudin 5 heterodimerisation partner. NHU cell differentiation was accompanied by relocalisation of TJ proteins to intercellular junctions. The differentiation-associated development of TJ formation in vitro reflected the stage-related TJ expression seen in situ. This was distinct from changes in TJ composition of NHU cells mediated by increasing the calcium concentration of the medium. Our results imply a role for PPARgamma and EGFR signalling pathways in regulating TJ formation in NHU cells and support the hypothesis that TJ development is an integral part of the urothelial differentiation programme.

Urothelial differentiation in chronically urine-deprived bladders of patients with end-stage renal disease.

BACKGROUND: It is unknown whether normal bladder voiding function, or soluble factors present in urine, contribute to the maturation and maintenance of the differentiated state of the uroepithelial cell lining of the lower urinary tract. METHODS: We used the urothelium of anuric patients on long-term hemodialysis, sampled at the time of renal transplantation, to investigate the expression of urothelial differentiation-associated antigens, including uroplakins (UPIa, UPIb, UPII, and UPIIIa), cytokeratin isotypes (CK7, CK8, CK13, CK14, CK17, CK18, and CK20), nuclear hormone receptors [peroxisome proliferators activated receptor-gamma (PPAR-gamma) and retinoid X receptor-alpha (RXR-alpha)], and a cell cycle marker (Ki-67). To determine whether urinary metabolites of the arachidonic pathway could induce urothelial differentiation, cultured normal human urothelial (NHU) cells were treated with 15-deoxy-delta12, 14-prostaglandin J2 (15d-PGJ2) and prostaglandin J2 (PGJ2). The expression levels of the markers of differentiation, the uroplakins, were assessed by ribonuclease protection assay. Results. When compared in a blinded analysis against control normal urothelium, no significant changes were found in the expression or localization patterns of any of the antigens studied in the anuric patients. Furthermore, neither 15d-PGJ2 nor PGJ2 were able to induce expression of the UPII gene in NHU cells, in contrast to cultures exposed to the pharmacologic PPAR-gamma agonist, troglitazone. Conclusion. These data provide prima facie evidence that exogenous urine-derived factors do not modulate the differentiation program in urothelium, suggesting that other urothelial- or serum-derived factors are likely to be involved. These findings are important in understanding post-developmental maturation and functional relationships in urologic tissues of the adult organism.

Role of PPARgamma and EGFR signalling in the urothelial terminal differentiation programme.

Recently, considerable interest has focused on the ability of activated peroxisome proliferator-activated receptor gamma (PPARgamma) to promote cytodifferentiation in adipocytes and some carcinoma cells; however, the role of PPARgamma in normal epithelial cytodifferentiation is unknown. Using uroplakin (UP) gene expression as a specific correlate of terminal urothelial cytodifferentiation, we investigated the differentiation-inducing effects of PPARgamma activation in normal human urothelial (NHU) cells grown as finite cell lines in monoculture. Two high-affinity activators of PPARgamma, troglitazone (TZ) and rosiglitazone (RZ) induced the expression of mRNA for UPII and UPIb and, to a lesser extent, UPIa. The specificity of the effect was shown by pretreating cells with a PPARgamma antagonist, GW9662, which attenuated the TZ-induced response in a dose-specific manner. The PPARgamma-mediated effect on UP gene expression was maximal when there was concurrent inhibition of autocrine-activated epidermal growth factor receptor (EGFR) signalling through either the phosphatidylinositol 3-kinase or extracellular signal-regulated kinase (ERK) pathways. The use of a specific EGFR tyrosine kinase inhibitor, PD153035, correlated with PPARgamma dephosphorylation and translocation to the nucleus, indicating a mechanism for regulating the balance between proliferation and differentiation. This is the first identification of specific factors involved in regulating differentiation-associated gene changes in urothelium and the first unambiguous evidence of a role for PPARgamma signalling in the terminal differentiation programme of a normal epithelium.

Regulation of the C-X-C chemokine, mob-1, gene expression in primary rat hepatocytes.

The chemokine, mob-1, is involved in inflammatory and immune responses and may be an important mediator of the inflammatory response in the liver. Here, we investigated the upstream signal pathways that could be involved in the regulation of mob-1 expression. We have found that in primary rat hepatocytes the isolation and subsequent culture of these cells induced mob-1 expression. A similar induction of mob-1 mRNA was observed when the hepatocytes were stimulated with interferon-gamma (IFN-gamma). When hepatocytes were stimulated with IFN-gamma or cytokine mixture (IFN-gamma, interleukin-1beta and tumour necrosis factor-alpha), c-Jun N-terminal kinase (JNK), p38 and extracellular-regulated kinase (ERK) were phosphorylated, suggesting an involvement of the mitogen-activated protein kinases (MAPK) in the induction of mob-1 expression. The p38 kinase inhibitor, SB 203580, and the NF-kappaB inhibitor, MG-132, inhibited the induction of mob-1 mRNA and the effects were not additive. These results demonstrate that in primary rat hepatocytes the transient induction of mob-1 expression was regulated by p38 kinase and NF-kappaB through a common regulatory pathway.