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High grade papillary urothelial carcinomas (pT1 or pT2). (a) Uroplakin (UP) labelling (red) is negative. (b) Scanning electron microscopy (SEM) shows that superficial urothelial cells are small and polymorphic. They have microvilli on their apical surface. (c) Antibody against P2X3 weakly labels (green) urothelial cells. (d) P2X5 labelling (red) is present in all urothelial cells. Nuclei of some cells are also labelled. (e) TRPV1 labelling is negative. (f) Weak labelling of TRPV4 in urothelial cells (U) is seen, but strong TRPV4 labelling (arrows) is seen in the compartments of the lamina propria (LP). In images (a) and (c)–(f), nuclei are labelled blue with DAPI. L = lumen. Scale bars = 50 μm.
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Terminal differentiation of urothelium is a prerequisite for blood-urine barrier formation and enables normal sensory function of the urinary bladder. In this study, urothelial differentiation of normal human urothelium and of low and high grade papillary urothelial carcinomas was correlated with the expression and localization of purinergic recept...
Citations
... It may also be that in the lungs, the ability of TRPV4 to sense mechanical changes in the cell microenvironment is essential for the morphogenesis of all the tissue compartments within the lungs, including epithelium, smooth muscle layer and the vascular compartment 75 . Furthermore, TRPV4 plays a role in the normal differentiation of corneal epithelium, urothelium and kidney epithelium 30,76,77 . Mechanosensitive features of TRPV4 are additionally needed for the differentiation and activation of immune cells 66,78 . ...
Biophysical cues from the cell microenvironment are detected by mechanosensitive components at the cell surface. Such machineries convert physical information into biochemical signaling cascades within cells, subsequently leading to various cellular responses in a stimulus-dependent manner. At the surface of extracellular environment and cell cytoplasm exist several ion channel families that are activated by mechanical signals to direct intracellular events. One of such channel is formed by transient receptor potential cation channel subfamily V member, TRPV4 that is known to act as a mechanosensor in wide variaty of tissues and control ion-influx in a spatio-temporal way. Here we report that TRPV4 is prominently expressed in the stem/progenitor cell populations of the mammary epithelium and seems important for the lineage-specific differentiation, consequently affecting mechanical features of the mature mammary epithelium. This was evident by the lack of several markers for mature myoepithelial and luminal epithelial cells in TRPV4-depleted cell lines. Interestingly, TRPV4 expression is controlled in a tension-dependent manner and it also impacts differentation process dependently on the stiffness of the microenvironment. Furthermore, such cells in a 3D compartment were disabled to maintain normal mammosphere structures and displayed abnormal lumen formation, size of the structures and disrupted cellular junctions. Mechanosensitive TRPV4 channel therefore act as critical player in the homeostasis of normal mammary epithelium through sensing the physical environment and guiding accordingly differentiation and structural organization of the bilayered mammary epithelium.
... Bladder cancer TRPV1 protein is easily detectable in the normal urothelium (Figure 8a). In noninvasive papillary urothelial carcinoma, TRPV1 expression is reduced (but still detectable) compared to normal urothelium [191], whereas in invasive urothelial carcinoma, TRPV1 staining is virtually absent (Figure 8b) [192,193]. According to these observations, TRPV1 immunostaining may help distinguish between non-invasive and invasive urothelial carcinoma. ...
... TRPV1 protein is easily detectable in the normal urothelium (Figure 8a). In n vasive papillary urothelial carcinoma, TRPV1 expression is reduced (but still detec compared to normal urothelium [191], whereas in invasive urothelial carcinoma, T staining is virtually absent (Figure 8b) [192,193]. According to these observations, T immunostaining may help distinguish between non-invasive and invasive urothelia cinoma. ...
... For example, TRPV1 is expressed in normal or inflamed gastric mucosa, but is absent in gastric adenocarcinoma [201]. Furthermore, TRPV1 is expressed both in the normal urothelium [254] and non-invasive papillary urothelial carcinoma [191]. By contrast, no TRPV1 expression was seen in invasive urothelial carcinoma [192,193]. ...
Temperature-sensitive transient receptor potential (TRP) channels (so-called “thermoTRPs”) are multifunctional signaling molecules with important roles in cell growth and differentiation. Several “thermoTRP” channels show altered expression in cancers, though it is unclear if this is a cause or consequence of the disease. Regardless of the underlying pathology, this altered expression may potentially be used for cancer diagnosis and prognostication. “ThermoTRP” expression may distinguish between benign and malignant lesions. For example, TRPV1 is expressed in benign gastric mucosa, but is absent in gastric adenocarcinoma. TRPV1 is also expressed both in normal urothelia and non-invasive papillary urothelial carcinoma, but no TRPV1 expression has been seen in invasive urothelial carcinoma. “ThermoTRP” expression can also be used to predict clinical outcomes. For instance, in prostate cancer, TRPM8 expression predicts aggressive behavior with early metastatic disease. Furthermore, TRPV1 expression can dissect a subset of pulmonary adenocarcinoma patients with bad prognosis and resistance to a number of commonly used chemotherapeutic agents. This review will explore the current state of this rapidly evolving field with special emphasis on immunostains that can already be added to the armoire of diagnostic pathologists.
... TRPs are involved in multiple processes, including pain [151], thermosensation (TRPV1 > 43 • C, TRPM8 cold) [152,153], low extracellular pH [153], axon survival (TRPV4) [154], stem cell fibrillar collagen assembly [29], blood pressure regulation [155], energy homeostasis [156], modulation of autophagy and proteasome activity [157], reciprocal crosstalk between the sensory nervous and immune systems [158], regulation of diet-induced obesity, insulin and leptin resistance [159], cancer [160,161], the development of severe bronchial asthma [162], and even in itch and inflammation [163]. ...
Focal vibration therapy seeks to restore the physiological function of tissues and the nervous system. Recommendations for vibration settings, e.g., that could improve residual limb health and prosthesis acceptance in people with amputation, are pending. To establish a physiological connection between focal vibration settings, clinical outcomes, and molecular and neuronal mechanisms, we combined the literature on focal vibration therapy, vibrotactile feedback, mechanosensitive Piezo ion channels, touch, proprioception, neuromodulation, and the recovery of blood vessels and nerves. In summary, intermittent focal vibration increases endothelial shear stress when applied superficially to blood vessels and tissues and triggers Piezo1 signaling, supporting the repair and formation of blood vessels and nerves. Conversely, stimulating Piezo1 in peripheral axon growth cones could reduce the growth of painful neuromas. Vibrotactile feedback also creates sensory inputs to the motor cortex, predominantly through Piezo2-related channels, and modulates sensory signals in the dorsal horn and ascending arousal system. Thus, sensory feedback supports physiological recovery from maladaptations and can alleviate phantom pain and promote body awareness and physical activity. We recommend focal vibration of phantom limb maps with frequencies from ~60–120 Hz and amplitudes up to 1 mm to positively affect motor control, locomotion, pain, nerves, and blood vessels while avoiding adverse effects.
... The results showed that, in the dry mouth group, K8 expression was significantly downregulated in the cells, whereas TRPV1 was robustly activated; moreover, P2X3 expression was upregulated at the ends of NFs. P2X3 belongs to the ATP-dependent purinoceptor family; it functions as a ligand-gated ion channel and transduces signals due to ATP-evoked nociception [41][42][43]. The results suggested that dry mouth induced the expression of nociceptors, such as P2X3, and triggered NF retraction, thereby activating TRPV1 in neuroepithelial cells in the taste buds. ...
Taste bud cell differentiation is extremely important for taste sensation. Immature taste bud cells cannot function during taste perception transmission to the nerve. In this study, we investigated whether hedgehog signaling affected taste bud cell differentiation and whether transient receptor potential vanilloid 1 (TRPV1) played a key role in dry mouth. The induction of dry mouth due to salivary gland resection (SGR) was confirmed on the basis of reduced salivation and disrupted fungiform papillae. The expression of keratin 8 (K8) of taste bud cells, neurofilament (NF), sonic hedgehog (Shh), and glioma-associated oncogene homolog 1 (Gli1) around taste bud cells was downregulated; however, the expression of TRPV1, P2X purinoceptor 3 (P2X3), and hematopoietic stem cell factor (c-Kit) was upregulated at the NF ends in the dry mouth group. To investigate the effect of TRPV1 defect on dry mouth, we induced dry mouth in the TRPV-/- group. The K8, NF, and P2X3 expression patterns were the same in the TRPV1 wild-type and TRPV1-/- dry mouth groups. However, Shh and c-Kit expression decreased regardless of dry mouth in the case of TRPV1 deficiency. These results indicated that TRPV1 positively regulated proliferation during taste bud cell injury by blocking the Shh/Gli1 pathway. In addition, not only cell proliferation but also differentiation of taste bud cells could not be regulated under TRPV1-deficiency conditions. Thus, TRPV1 positively regulates taste bud cell innervation and differentiation; this finding could be valuable in the clinical treatment of dry mouth-related taste dysfunction.
... Western blotting and IHC analysis showed that P2X3 receptor protein expression is similar in normal urothelium (n = 13) and low-grade papillary carcinoma (n = 12), but decreased in high-grade papillary carcinoma (n = 6) of the human bladder. [85] In contrast, P2X5 receptor protein expression was present in normal urothelium and high-grade carcinoma but was diminished in low-grade carcinoma. [85] The authors propose that P2X3 correlates with urothelial differentiation and might be involved in high-grade papillary carcinoma pathogenesis, while P2X5 does not. ...
... [85] In contrast, P2X5 receptor protein expression was present in normal urothelium and high-grade carcinoma but was diminished in low-grade carcinoma. [85] The authors propose that P2X3 correlates with urothelial differentiation and might be involved in high-grade papillary carcinoma pathogenesis, while P2X5 does not. In another study, P2Y2 enhancer RNA (P2Y2e) was overexpressed in 29 of 38 bladder cancer patient cancer tissues compared to paracancerous tissues. ...
P2 purinergic receptors are involved in the normal function of the kidney, bladder, and prostate via signaling that occurs in response to extracellular nucleotides. Dysregulation of these receptors is common in pathological states and often associated with disease initiation, progression, or aggressiveness. Indeed, P2 purinergic receptor expression is altered across multiple urologic disorders including chronic kidney disease, polycystic kidney disease, interstitial cystitis, urinary incontinence, overactive bladder syndrome, prostatitis, and benign prostatic hyperplasia. P2 purinergic receptors are likewise indirectly associated with these disorders via receptor-mediated inflammation and pain, a common characteristic across most urologic disorders. Furthermore, select P2 purinergic receptors are overexpressed in urologic cancer including renal cell carcinoma, urothelial carcinoma, and prostate adenocarcinoma, and pre-clinical studies depict P2 purinergic receptors as potential therapeutic targets. Herein, we highlight the compelling evidence for the exploration of P2 purinergic receptors as biomarkers and therapeutic targets in urologic cancers and other urologic disease. Likewise, there is currently optimism for P2 purinergic receptor-targeted therapeutics for the treatment of inflammation and pain associated with urologic diseases. Further exploration of the common pathways linking P2 purinergic receptor dysregulation to urologic disease might ultimately help in gaining new mechanistic insight into disease processes and therapeutic targeting.
... 77 Several studies showed altered expression of TRPV1 in patients with BPS as well as in animal models, but until now only one study demonstrated this by IF. 74,75,78,79 On the other hand, there is more consistency about TRPV4 localization in the normal urothelium, particularly in the basal cell layer. 76,80,81 IEM further showed that TRPV4 is localized near the basal plasma membranes adjacent to the basal lamina. 73 Although some studies reported on important role of TRPV4 in BPS, 82,83 no IHC confirmation is available, yet. ...
The urothelium, an epithelium of the urinary bladder, primarily functions as blood-urine permeability barrier. The urothelium has a very slow turn-over under normal conditions but is capable of extremely fast response to injury. During regeneration urothelium either restores normal function or undergoes altered differentiation pathways, the latter being the cause of several bladder diseases. In this review, we describe the structure of the apical plasma membrane that enables barrier function, the role of urothelium specific proteins uroplakins and the machinery for polarized membrane transports in terminally differentiated superficial umbrella cells. We address key markers, such as keratins, cancer stem cell markers, retinoic acid signalling pathway proteins and transient receptor potential channels and purinergic receptors that drive normal and altered differentiation in bladder cancer and bladder pain syndrome. Finally, we discuss uncertainties regarding research, diagnosis and treatment of bladder pain syndrome. Throughout the review, we emphasise the contribution of immunohistochemistry in advancing our understanding of processes in normal and diseased bladder as well as the most promising possibilities for improved bladder cancer and bladder pain syndrome management.
... Although TRPV1 expression on functionally-relevant levels in urothelium still remains the matter of controversy 26 , in human urothelial cancer specimens TRPV1 mRNA and protein expression is, in fact, progressively decreasing from low grade to high-grade muscle-invasive urothelial carcinoma as compared to normal urothelium 44,45 . Thus, TRPV1 may play dual role in bladder cancer: on the one hand, the decrease of its urothelial content would promote oncogenic apoptosis resistance 46 and, on the other hand, its enhanced function as a receptor of various sensory modalities in bladder afferents would potentiate DSM contractility. ...
Urinary incontinence of idiopathic nature is a common complication of bladder cancer, yet, the mechanisms underlying changes in bladder contractility associated with cancer are not known. Here by using tensiometry on detrusor smooth muscle (DSM) strips from normal rats and rats with bladder cancer induced by known urothelial carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), we show that bladder cancer is associated with considerable changes in DSM contractility. These changes include: (1) decrease in the amplitude and frequency of spontaneous contractions, consistent with the decline of luminal pressures during filling, and detrusor underactivity; (2) diminution of parasympathetic DSM stimulation mainly at the expense of m-cholinergic excitatory transmission, suggestive of difficulty in bladder emptying and weakening of urine stream; (3) strengthening of TRPV1-dependent afferent limb of micturition reflex and TRPV1-mediated local contractility, promoting urge incontinence; (4) attenuation of stretch-dependent, TRPV4-mediated spontaneous contractility leading to overflow incontinence. These changes are consistent with the symptomatic of bladder dysfunction in bladder cancer patients. Considering that BBN-induced urothelial lesions in rodents largely resemble human urothelial lesions at least in their morphology, our studies establish for the first time underlying reasons for bladder dysfunction in bladder cancer.
... However, some studies on P2X5R in other cancer forms are published. P2X5 receptors have been identified in squamous cell carcinomas of the skin and prostate cancers and different grades of papillary urothelial carcinoma [122][123][124]. For more complete reviews on P2XR and other cancers refer to [4,125]. ...
The purinergic signaling has an important role in regulating pancreatic exocrine secretion. The exocrine pancreas is also a site of one of the most serious cancer forms, the pancreatic ductal adenocarcinoma (PDAC). Here, we explore how the network of purinergic and adenosine receptors, as well as ecto-nucleotidases regulate normal pancreatic cells and various cells within the pancreatic tumor microenvironment. In particular, we focus on the P2X7 receptor, P2Y2 and P2Y12 receptors, as well as A2 receptors and ecto-nucleotidases CD39 and CD73. Recent studies indicate that targeting one or more of these candidates could present new therapeutic approaches to treat pancreatic cancer. In pancreatic cancer, as much as possible of normal pancreatic function should be preserved, and therefore physiology of purinergic signaling in pancreas needs to be considered.
... TRPV4 is known to play an important role in tumour cell proliferation, differentiation and metastasis (Yu et al. 2019), but it does not seem to be involved in urothelial proliferation and cancer (Kalogris et al. 2010;Sterle et al. 2014). However, studies on TRPV4 channelopathies have identified a potential link between TRPV4 and overactive bladder (Nilius & Voets, 2013). ...
The transient receptor potential vanilloid type 4, TRPV4, is a polymodal cation channel which can be activated by diverse stimuli including mechanical, thermal and chemical cues. In the urinary bladder, TRPV4 is not only abundantly expressed in the urothelium but may also be localized in subepithelium, detrusor smooth muscles and afferent neurons. Emerging evidence indicates that the TRPV4 channel plays a sensory role in the uroepithelium, where it may regulate the release of sensory mediators such as ATP, which in turn modulates afferent nerve activity in response to bladder filling during the urination cycle. TRPV4 may also directly regulate detrusor contractility and the urothelial barrier function. Altered TRPV4 expression has been detected in various pathological bladder conditions. As such, TRPV4 may be a promising therapeutic target for bladder dysfunctions. image
... IHC was performed as previously described. 26,27 Briefly, all sections were incubated in 2.5% bovine serum albumin (BSA) in PBS for 1 h at room temperature (RT) to block nonspecific labelling. Then, sections were incubated overnight at 4°C with primary antibodies (pAb) anti-UPs antibodies (diluted 1:5000 for paraffin sections and 1:10000 for cryo-semithin sections). ...
Lectin histochemistry (LHC) and immunohistochemistry (IHC), which demonstrate the composition and localisation of sugar residues and proteins in cell membranes, respectively, are generally used separately. Using these two methods, we previously demonstrated that malignant transformation of urothelial cells results in the alterations of protein glycosylation and reduced expression of urothelium-specific integral membrane proteins uroplakins (UPs). However, the correlation between these changes was not studied yet. To evaluate this correlation, we developed innovative method, which we named combined lectin- and immuno- histochemistry (CLIH). We used human biopsies of 6 normal urothelia and 9 papillary urothelial carcinomas, i.e. 3 papillary urothelial neoplasms of low malignant potential (PUNLMP), 3 non-invasive papillary urothelial carcinomas of low grade (pTa, l.g.), and 3 invasive papillary urothelial carcinomas of high grade (pT1, h.g.). We tested five different protocols (numbered 1-5) of CLIH on paraffin and cryo-semithin sections and compared them with LHC and IHC performed separately. Additionally, we carried out western and lectin blotting with antibodies against UPs and lectins Amaranthus caudatus agglutinin (ACA), Datura stramonium agglutinin (DSA), and jacalin, respectively. We showed that incubation with primary antibodies first, followed by the mixture of secondary antibodies and lectins is the most efficient CLIH method (protocol number 5). Additionally, 300 nm thick cryo-semithin sections enabled better resolution of co-localisation between sugar residues and proteins than 5 µm thick paraffin sections. In the normal urothelium, CLIH showed co-localisation of lectins ACA and jacalin with UPs in the apical plasma membrane (PM) of superficial umbrella cells. In papillary urothelial carcinomas, all three lectins (ACA, DSA and jacalin) labelled regions of apical PM, where they occasionally co-localised with UPs. Western and lectin blotting confirmed the differences between normal urothelium and papillary urothelial carcinomas. Our results show that CLIH, when used with various sets of lectins and antigens, is a useful, quick, and reliable method that could be applied for basic cell biology research as well as detailed subtyping of human urothelial carcinomas.
