Cellular and Molecular Gastroenterology and Hepatology
Xu H, Li J, Chen H, Ghishan FK.
PMID: - | DOI: 10.1016/j.jcmgh.2018.08.005
Abstract
Background and Aims
Lgr5 overexpression has been detected in colorectal cancers (CRCs), including some cases of colitis-associated CRCs. In colitis-associated CRCs, chronic inflammation is a contributing factor in carcinogenesis. We recently reported that intestinal sodium/hydrogen exchanger isoform 8 (NHE8) plays an important role in intestinal mucosal protection and that loss of NHE8 expression results in ulcerative colitis (UC)-like condition. Therefore, we hypothesized that NHE8 may be involved in the development of intestinal tumors.
Methods
We assessed NHE8 expression in human CRCs by IHC and studied tumor burden in NHE8KO mice using an AOM/DSS colon cancer model. We also evaluated cell proliferation in HT29NHE8KO cells and assessed tumor growth in NSG mice xenografted with HT29NHE8KO cells. To verify if a relationship exists between Lgr5 and NHE8 expression, we analyzed Lgr5 expression in NHE8KO mice by PCR and in situ hybridization. Lgr5 expression and cell proliferation in the absence of NHE8 were confirmed in colonic organoid cultures. The expression of β-catenin and c-Myc were also analyzed to evaluate Wnt/β-catenin activation.
Results
NHE8 was undetectable in human CRC tissues. Whereas only 9% of NHE8WT mice exhibited tumorigenesis in the AOM/DSS colon cancer model, almost ten times more NHE8KO mice (89%) developed tumors. In the absence of NHE8, a higher colony formation unit was discovered in HT29NHE8KO cells. In NSG mice, larger tumors developed at the site where HT29NHE8KO cells were injected compared to HT29NHE8WT cells. Furthermore, NHE8 deficiency resulted in elevated Lgr5 expression in the colon, in HT29 derived tumors, and in colonoids. The absence of NHE8 also increased Wnt/β-catenin activation.
Conclusions
NHE8 might be an intrinsic factor that regulates Wnt/β-catenin in the intestine.
Expression of LGR5 in mammary myoepithelial cells and in triple-negative breast cancers
Lee, HJ;Myung, JK;Kim, HS;Lee, DH;Go, HS;Choi, JH;Koh, HM;Lee, SJ;Jang, B;
PMID: 34493772 | DOI: 10.1038/s41598-021-97351-y
Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no β-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted.
Buhidma, Y;Hobbs, C;Malcangio, M;Duty, S;
PMID: 37100804 | DOI: 10.1038/s41531-023-00510-3
Pain is a key non-motor feature of Parkinson's disease (PD) that significantly impacts on life quality. The mechanisms underlying chronic pain in PD are poorly understood, hence the lack of effective treatments. Using the 6-hydroxydopamine (6-OHDA) lesioned rat model of PD, we identified reductions in dopaminergic neurons in the periaqueductal grey (PAG) and Met-enkephalin in the dorsal horn of the spinal cord that were validated in human PD tissue samples. Pharmacological activation of D1-like receptors in the PAG, identified as the DRD5+ phenotype located on glutamatergic neurons, alleviated the mechanical hypersensitivity seen in the Parkinsonian model. Downstream activity in serotonergic neurons in the Raphé magnus (RMg) was also reduced in 6-OHDA lesioned rats, as detected by diminished c-FOS positivity. Furthermore, we identified increased pre-aggregate α-synuclein, coupled with elevated activated microglia in the dorsal horn of the spinal cord in those people that experienced PD-related pain in life. Our findings have outlined pathological pathways involved in the manifestation of pain in PD that may present targets for improved analgesia in people with PD.
Brain Struct Funct. 2015 Jul 10.
Hackett TA, Clause AR, Takahata T, Hackett NJ, Polley DB.
PMID: 26159773
Vesicular transporter proteins are an essential component of the presynaptic machinery that regulates neurotransmitter storage and release. They also provide a key point of control for homeostatic signaling pathways that maintain balanced excitation and inhibition following changes in activity levels, including the onset of sensory experience. To advance understanding of their roles in the developing auditory forebrain, we tracked the expression of the vesicular transporters of glutamate (VGluT1, VGluT2) and GABA (VGAT) in primary auditory cortex (A1) and medial geniculate body (MGB) of developing mice (P7, P11, P14, P21, adult) before and after ear canal opening (~P11-P13). RNA sequencing, in situ hybridization, and immunohistochemistry were combined to track changes in transporter expression and document regional patterns of transcript and protein localization. Overall, vesicular transporter expression changed the most between P7 and P21. The expression patterns and maturational trajectories of each marker varied by brain region, cortical layer, and MGB subdivision. VGluT1 expression was highest in A1, moderate in MGB, and increased with age in both regions. VGluT2 mRNA levels were low in A1 at all ages, but high in MGB, where adult levels were reached by P14. VGluT2 immunoreactivity was prominent in both regions. VGluT1 + and VGluT2 + transcripts were co-expressed in MGB and A1 somata, but co-localization of immunoreactive puncta was not detected. In A1, VGAT mRNA levels were relatively stable from P7 to adult, while immunoreactivity increased steadily. VGAT + transcripts were rare in MGB neurons, whereas VGAT immunoreactivity was robust at all ages. Morphological changes in immunoreactive puncta were found in two regions after ear canal opening. In the ventral MGB, a decrease in VGluT2 puncta density was accompanied by an increase in puncta size. In A1, perisomatic VGAT and VGluT1 terminals became prominent around the neuronal somata. Overall, the observed changes in gene and protein expression, regional architecture, and morphology relate to-and to some extent may enable-the emergence of mature sound-evoked activity patterns. In that regard, the findings of this study expand our understanding of the presynaptic mechanisms that regulate critical period formation associated with experience-dependent refinement of sound processing in auditory forebrain circuits.
European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology
Ngoc, KH;Kecskés, A;Kepe, E;Nabi, L;Keeble, J;Borbély, É;Helyes, Z;
PMID: 37156112 | DOI: 10.1016/j.euroneuro.2023.04.017
The Transient Receptor Potential Vanilloid 1 (TRPV1) non-selective cation channel predominantly expressed in primary sensory neurons of the dorsal root and trigeminal ganglia mediates pain and neurogenic inflammation. TRPV1 mRNA and immunoreactivity were described in the central nervous system (CNS), but its precise expression pattern and function have not been clarified. Here we investigated Trpv1 mRNA expression in the mouse brain using ultrasensitive RNAScope in situ hybridization. The role of TRPV1 in anxiety, depression-like behaviors and memory functions was investigated by TRPV1-deficient mice and pharmacological antagonism by AMG9810. Trpv1 mRNA is selectively expressed in the supramammillary nucleus (SuM) co-localized with Vglut2 mRNA, but not with tyrosine hydroxylase immunopositivity demonstrating its presence in glutamatergic, but not dopaminergic neurons. TRPV1-deleted mice exhibited significantly reduced anxiety in the Light-Dark box and depression-like behaviors in the Forced Swim Test, but their performance in the Elevated Plus Maze as well as their spontaneous locomotor activity, memory and learning function in the Radial Arm Maze, Y-maze and Novel Object Recognition test were not different from WTs. AMG9810 (intraperitoneal injection 50 mg/kg) induced anti-depressant, but not anxiolytic effects. It is concluded that TRPV1 in the SuM might have functional relevance in mood regulation and TRPV1 antagonism could be a novel perspective for anti-depressant drugs.
Nakajima T, Uehara T, Maruyama Y, Iwaya M, Kobayashi Y, Ota H.
PMID: 27593551 | DOI: 10.1111/pin.12451
Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is a putative intestinal stem cell marker that is also expressed in various tumors. To analyze its pathological characteristics in mucosal gastric signet-ring cell carcinoma (SRCC), we investigated Lgr5 expression in 35 intramucosal gastric SRCC patients using RNAscope, a newly developed RNA in situ hybridization technique. Lgr5 expression in individual tumor cells was scored semi-quantitatively from 0 to 400. Ki67 was also examined by immunohistochemistry, with a linear arrangement of Ki67-expressing cells present in 20 of 35 cases. This area of Ki67-expressing cells was topographically divided into upper, middle, and lower regions. All cases with linear Ki67 expression patterns also had Lgr5-positive cells arranged in a linear fashion in the lower area-which was distinct from the area of high Ki67 expression. The rate of Ki67 positivity in Lgr5-positive cells was significantly lower than that of Lgr5-negative cells in areas of high Ki67 expression. In intramucosal SRCC, the low mitotic activity of Lgr5-positive cells suggests that they may represent cancer stem cells as seen in other types of stomach carcinomas. Intramucosal SRCC may therefore contain stem cells expressing Lgr5 in the lower area of the lamina propria, akin to normal gastric pyloric mucosa.
Kamakura, M;Uehara, T;Iwaya, M;Asaka, S;Kobayashi, S;Nakajima, T;Kinugawa, Y;Nagaya, T;Yoshizawa, T;Shimizu, A;Ota, H;Umemura, T;
PMID: 35123536 | DOI: 10.1186/s13000-022-01203-w
Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a strong cancer stem cell marker in colorectal cancer; however, there are many unclear aspects of LGR5 expression in pancreatic cancer. It has been reported that the interaction between tumor cells and stroma at the fat infiltration site has a significant effect on pancreatic cancer prognosis. Therefore, we report a clinicopathological study of LGR5 expression at the fat invasion front in pancreatic cancer.LGR5 expression was analyzed in 40 pancreatic ductal adenocarcinoma cases with RNAscope, which is a newly developed high-sensitivity in situ hybridization method. Epithelial-mesenchymal transition (EMT) was analyzed by the expression of E-cadherin and vimentin via immunohistochemistry.LGR5-positive dots were identified in all cases, especially with glandular formation. In the fat invasion front, a high histological grade showed significantly reduced LGR5 expression compared with a low histological grade (p=0.0126). LGR5 expression was significantly higher in the non-EMT phenotype group than in EMT phenotype group (p=0.0003). Additionally, LGR5 expression was significantly lower in cases with high vascular invasion than in those with low vascular invasion (p=0.0244).These findings suggest that decreased LGR5 expression in the fat invasion front is associated with more aggressive biological behavior in pancreatic ductal adenocarcinoma, with higher tumor grade, EMT phenotype, and higher vascular invasion.
Krajewski-Hall SJ, Miranda Dos Santos F, McMullen NT, Blackmore EM, Rance NE.
PMID: 30753503 | DOI: 10.1210/en.2018-00934
We have proposed that KNDy (kisspeptin/neurokinin B/dynorphin) neurons contribute to hot flushes via projections to neurokinin 3 receptor (NK3R) expressing neurons in the median preoptic nucleus (MnPO). To characterize the thermoregulatory role of MnPO NK3R neurons in female mice, we ablated these neurons using injections of saporin toxin conjugated to a selective NK3R agonist. Loss of MnPO NK3R neurons increased core temperature (TCORE) during the light phase, with frequency distributions indicating a regulated shift in the balance point. The rise in TCORE in ablated mice occurred despite changes in ambient temperature (TAMBIENT) and regardless of estrogen status. We next determined if an acute increase in TAMBIENT or higher TCORE would induce Fos in preoptic EGFP-immunoreactive neurons in Tacr3-EGFP mice. Fos-activation was increased in the MnPO, but there was no induction of Fos in NK3R (EGFP-immunoreactive) neurons. Thus, MnPO NK3R neurons are not activated by warm thermosensors in the skin or viscera and are not warm-sensitive neurons. Finally, RNAscope was used to determine if Tacr3 (NK3R) mRNA was co-expressed with VGLUT2 or VGAT mRNA, markers of glutamatergic or GABAergic neurotransmission, respectively. Interestingly, 94% of NK3R neurons in the MnPO were glutamatergic, whereas in the adjacent MPA, 97% of NK3R neurons were GABAergic. Thus, NK3R neurons in the MnPO are glutamatergic and play a role in reducing TCORE, but they are not activated by warm thermal stimuli (internal or external). These studies suggest that KNDy neurons modulate thermosensory pathways for heat-defense indirectly, via a subpopulation of glutamatergic MnPO neurons that express NK3R.
Cooper, S;Souza, L;Worker, C;Gayban, A;Buller, S;Satou, R;Feng Earley, Y;
| DOI: 10.3390/biom12091169
The brain renin-angiotensin system plays important roles in blood pressure and cardiovascular regulation. There are two isoforms of prorenin in the brain: the classic secreted form (prorenin/sREN) encoded by renin-a, and an intracellular form (icREN) encoded by renin-b. Emerging evidence indicates the importance of renin-b in cardiovascular and metabolic regulation. However, the role of endogenous brain prorenin in the development of salt-sensitive hypertension remains undefined. In this study, we test the hypothesis that renin-a produced locally in the brain contributes to the pathogenesis of hypertension. Using RNAscope, we report for the first time that renin mRNA is expressed in several regions of the brain, including the subfornical organ (SFO), the paraventricular nucleus of the hypothalamus (PVN), and the brainstem, where it is found in glutamatergic, GABAergic, cholinergic, and tyrosine hydroxylase-positive neurons. Notably, we found that renin mRNA was significantly elevated in the SFO and PVN in a mouse model of DOCA-salt-induced hypertension. To examine the functional importance of renin-a in the SFO, we selectively ablated renin-a in the SFO in renin-a-floxed mice using a Cre-lox strategy. Importantly, renin-a ablation in the SFO attenuated the maintenance of DOCA-salt-induced hypertension and improved autonomic function without affecting fluid or sodium intake. Molecularly, ablation of renin-a prevented the DOCA-salt-induced elevation in NADPH oxidase 2 (NOX2) in the SFO without affecting NOX4 or angiotensin II type 1 and 2 receptors. Collectively, our findings demonstrate that endogenous renin-a within the SFO is important for the pathogenesis of salt-sensitive hypertension.
Hackett TA
PMID: 30315630 | DOI: 10.1002/ar.23907
In the brain, purines such as ATP and adenosine can function as neurotransmitters and co-transmitters, or serve as signals in neuron-glial interactions. In thalamocortical (TC) projections to sensory cortex, adenosine functions as a negative regulator of glutamate release via activation of the presynaptic adenosine A1 receptor (A1 R). In the auditory forebrain, restriction of A1 R-adenosine signaling in medial geniculate (MG) neurons is sufficient to extend LTP, LTD, and tonotopic map plasticity in adult mice for months beyond the critical period. Interfering with adenosine signaling in primary auditory cortex (A1) does not contribute to these forms of plasticity, suggesting regional differences in the roles of A1 R-mediated adenosine signaling in the forebrain. To advance understanding of the circuitry, in situ hybridization was used to localize neuronal and glial cell types in the auditory forebrain that express A1 R transcripts (Adora1), based on co-expression with cell-specific markers for neuronal and glial subtypes. In A1, Adora1 transcripts were concentrated in L3/4 and L6 of glutamatergic neurons. Subpopulations of GABAergic neurons, astrocytes, oligodendrocytes, and microglia expressed lower levels of Adora1. In MG, Adora1 was expressed by glutamatergic neurons in all divisions, and subpopulations of all glial classes. The collective findings imply that A1 R-mediated signaling broadly extends to all subdivisions of auditory cortex and MG. Selective expression by neuronal and glial subpopulations suggests that experimental manipulations of A1 R-adenosine signaling could impact several cell types, depending on their location. Strategies to target Adora1 in specific cell types can be developed from the data generated here.
Haidar M, Tin K, Zhang C, Nategh M, Covita J, Wykes AD, Rogers J and Gundlach AL
PMID: 30906254 | DOI: 10.3389/fnana.2019.00030
Relaxin-3 is a highly conserved neuropeptide abundantly expressed in neurons of the nucleus incertus (NI), which project to nodes of the septohippocampal system (SHS) including the medial septum/diagonal band of Broca (MS/DB) and dorsal hippocampus, as well as to limbic circuits. High densities of the Gi/o-protein-coupled receptor for relaxin-3, known as relaxin-family peptide-3 receptor (RXFP3) are expressed throughout the SHS, further suggesting a role for relaxin-3/RXFP3 signaling in modulating learning and memory processes that occur within these networks. Therefore, this study sought to gain further anatomical and functional insights into relaxin-3/RXFP3 signaling in the mouse MS/DB. Using Cre/LoxP recombination methods, we assessed locomotion, exploratory behavior, and spatial learning and long-term reference memory in adult C57BL/6J Rxfp3 (loxP/loxP) mice with targeted depletion of Rxfp3 in the MS/DB. Following prior injection of an AAV((1/2))-Cre-IRES-eGFP vector into the MS/DB to delete/deplete Rxfp3 mRNA/RXFP3 protein, mice tested in a Morris water maze (MWM) displayed an impairment in allocentric spatial learning during acquisition, as well as an impairment in long-term reference memory on probe day. However, RXFP3-depleted and control mice displayed similar motor activity in a locomotor cell and exploratory behavior in a large open-field (LOF) test. A quantitative characterization using multiplex, fluorescent in situ hybridization (ISH) identified a high level of co-localization of Rxfp3 mRNA and vesicular GABA transporter (vGAT) mRNA in MS and DB neurons (~87% and ~95% co-expression, respectively). Rxfp3 mRNA was also detected, to a correspondingly lesser extent, in vesicular glutamate transporter 2 (vGlut2) mRNA-containing neurons in MS and DB (~13% and ~5% co-expression, respectively). Similarly, a qualitative assessment of the MS/DB region, identified Rxfp3 mRNA in neurons that expressed parvalbumin (PV) mRNA (reflecting hippocampally-projecting GABA neurons), whereas choline acetyltransferase mRNA-positive (acetylcholine) neurons lacked Rxfp3 mRNA. These data are consistent with a qualitative immunohistochemical analysis that revealed relaxin-3-immunoreactive nerve fibers in close apposition with PV-immunoreactive neurons in the MS/DB. Together these studies suggest relaxin-3/RXFP3 signaling in the MS/DB plays a role in modulating specific learning and long-term memory associated behaviors in adult mice via effects on GABAergic neuron populations known for their involvement in modulating hippocampal theta rhythm and associated cognitive processes.
LGR5-Expressing Cells in the Healing Process of Post-ESD Ulcers in Gastric Corpus
Digestive diseases and sciences
Tobe, Y;Uehara, T;Nakajima, T;Iwaya, M;Kobayashi, Y;Kinugawa, Y;Kuraishi, Y;Ota, H;
PMID: 34081250 | DOI: 10.1007/s10620-021-07059-2
LGR5 is a promising stem cell marker in gastric pylorus, but there are few reports on its expression in human gastric corpus.To investigate the involvement of LGR5 expression in gastric corpus ulcer regeneration in humans.LGR5 expression was analyzed in five post-ESD ulcers during the healing process of regenerating epithelial cells of the gastric corpus. LGR5 expression was detected by mRNA in situ hybridization using an RNA scope kit. Immunohistochemistry of MUC6, HIK1083, and pepsinogen 1 (PG1) was performed to identify cell differentiation.We defined MUC6+/HIK1083-/PG1-, MUC6+/HIK1083+/PG1-, MUC6+/HIK1083+/PG1+, MUC6+/HIK1083-/PG1+, and MUC6-/HIK1083-/PG1+cells as pseudopyloric mucosa (PPM) phase 1 (PPM1), PPM phase 2 (PPM2), PPM phase 3 (PPM3), immature chief cells (ICC), and mature chief cells (MCC) in order from the ulcer center, respectively. In the regenerated mucosa around post-ESD ulcers, LGR5 expression was observed throughout the gland in PPM1-PPM3, but it was limited to the bottom of the gland in ICC and MCC. Furthermore, LGR5 expression was not identified in the normal gastric corpus. The H-score of PPM2 was significantly higher than that of PPM3 (P = 0.0313). The H-score of PPM3 was significantly higher than that of ICC (P = 0.0313). The LGR5 H-score was higher at the immature stage, which decreased gradually with progression of the differentiation stage.LGR5 expression appears to contribute to mucosal regeneration in the human gastric corpus. The application of LGR5 expression analysis to mucosal regeneration and fundic gland-type gastric tumors is expected.
