Probes for INS

Abstract

STUDY QUESTION:

Is human endometrial leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) gene expression limited to the postulated epithelial stem cell niche, stratum basalis glands, and is it hormonally regulated?

SUMMARY ANSWER:

LGR5 expressing cells are not limited to the postulated stem cell niche but LGR5 expression is hormonally regulated.

WHAT IS KNOWN ALREADY:

The human endometrium is a highly regenerative tissue; however, endometrial epithelial stem cell markers are yet to be confirmed. LGR5 is a marker of stem cells in various epithelia.

STUDY DESIGN, SIZE, DURATION:

The study was conducted at a University Research Institute. Endometrial samples from 50 healthy women undergoing benign gynaecological surgery with no endometrial pathology at the Liverpool Women's hospital were included and analysed in the following six sub-categories; proliferative, secretory phases of menstrual cycle, postmenopausal, those using oral and local progestagens and samples for in vitro explant culture.

PARTICIPANTS/MATERIALS, SETTING, METHODS:

In this study, we used the gold standard method, in situ hybridisation (ISH) along with qPCR and a systems biology approach to study the location of LGR5 gene expression in full thickness human endometrium and Fallopian tubes. The progesterone regulation of endometrial LGR5 was examined in vivo and in short-term cultured endometrial tissue explants in vitro. LGR5 expression was correlated with epithelial proliferation (Ki67), and expression of previously reported epithelia progenitor markers (SOX9 and SSEA-1) immunohistochemistry (IHC).

MAIN RESULTS AND THE ROLE OF CHANCE:

LGR5 gene expression was significantly higher in the endometrial luminal epithelium than in all other epithelial compartments in the healthy human endometrium, including the endometrial stratum basalis (P < 0.05). The strongest SSEA-1 and SOX9 staining was observed in the stratum basalis glands, but the general trend of SOX9 and SSEA-1 expression followed the same cyclical pattern of expression as LGR5. Stratum functionalis epithelial Ki67-LI and LGR5 expression levels correlated significantly (r = 0.74, P = 0.01), however, they did not correlate in luminal and stratum basalis epithelium (r = 0.5 and 0.13, respectively). Endometrial LGR5 demonstrates a dynamic spatiotemporal expression pattern, suggesting hormonal regulation. Oral and local progestogens significantly reduced endometrial LGR5 mRNA levels compared with women not on hormonal treatment (P < 0.01). Our data were in agreement with in silico analysis of published endometrial microarrays.

LARGE SCALE DATA:

We did not generate our own large scale data but interrogated publically available large scale data sets.

LIMITATIONS, REASONS FOR CAUTION:

In the absence of reliable antibodies for human LGR5 protein and validated lineage markers for the various epithelial populations that potentially exist within the endometrium, our study does not formally characterise or examine the functional ability of the resident LGR5+ cells as multipotent.

WIDER IMPLICATIONS OF THE FINDINGS:

These data will facilitate future lineage tracing studies in the human endometrial epithelium; to identify the location of stem cells and further complement the in vitro functional studies, to confirm if the LGR5 expressing epithelial cells indeed represent the epithelial stem cell population.

Neurotransmitter switching in the adult mammalian brain occurs following photoperiod-induced stress, but the mechanism of regulation is unknown. Here, we demonstrate that elevated activity of dopaminergic neurons in the paraventricular nucleus of the hypothalamus (PaVN) in the adult rat is required for the loss of dopamine expression after long-day photoperiod exposure. The transmitter switch occurs exclusively in PaVN dopaminergic neurons that coexpress vesicular glutamate transporter 2 (VGLUT2), is accompanied by a loss of dopamine type 2 receptors (D2Rs) on corticotrophin-releasing factor (CRF) neurons, and can lead to increased release of CRF. Suppressing activity of all PaVN glutamatergic neurons decreases the number of inhibitory PaVN dopaminergic neurons, indicating homeostatic regulation of transmitter expression in the PaVN.

A subset of midbrain dopamine (DA) neurons express vesicular glutamate transporter 2 (VgluT2), which facilitates synaptic vesicle loading of glutamate. Recent studies indicate that such expression can modulate DA-dependent reward behaviors, but little is known about functional consequences of DA neuron VgluT2 expression in neurodegenerative diseases like Parkinson's disease (PD). Here, we report that selective deletion of VgluT2 in DA neurons in conditional VgluT2-KO (VgluT2-cKO) mice abolished glutamate release from DA neurons, reduced their expression of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB), and exacerbated the pathological effects of exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, viral rescue of VgluT2 expression in DA neurons of VglutT2-cKO mice restored BDNF/TrkB expression and attenuated MPTP-induced DA neuron loss and locomotor impairment. Together, these findings indicate that VgluT2 expression in DA neurons is neuroprotective. Genetic or environmental factors causing reduced expression or function of VgluT2 in DA neurons may place some individuals at increased risk for DA neuron degeneration. Therefore, maintaining physiological expression and function of VgluT2 in DA neurons may represent a valid molecular target for the development of preventive therapeutic interventions for PD.

Therapeutic strategies based on a specific oncogenic target are better justified when elimination of that particular oncogene reduces tumorigenesis in a model organism. One such oncogene, Musashi 1 (Msi-1), regulates translation of target mRNAs and is implicated in promoting tumorigenesis in colon and other tissues. Msi-1 targets include the tumor suppressor Adenomatous Polyposis Coli (Apc), a Wnt pathway antagonist lost in ∼80% of all colorectal cancers. Cell culture experiments have established that Msi-1 is a Wnt target, thus positioning Msi-1 and Apc as mutual antagonists in a double negative feedback loop. Here, we report that intestines from mice lacking Msi-1 display aberrant Apc/Msi-1 double negative feedback, reduced Wnt and Notch signaling, decreased proliferation, and changes in stem cell populations, features predicted to suppress tumorigenesis. Indeed, ApcMin and Apc1322T mice have a dramatic reduction in intestinal polyp number when Msi-1 is deleted. Together, these results provide genetic evidence that Msi-1 contributes to intestinal tumorigenesis driven by Apc loss, and validate the pursuit of Msi-1 inhibitors as chemo-prevention agents to reduce tumor burden.

Solid malignancies have been speculated to depend on cancer stem cells (CSCs) for expansion and relapse after therapy. Here we report on quantitative analyses of lineage tracing data from primary colon cancer xenograft tissue to assess CSC functionality in a human solid malignancy. The temporally obtained clone size distribution data support a model in which stem cell function in established cancers is not intrinsically, but is entirely spatiotemporally orchestrated. Functional stem cells that drive tumour expansion predominantly reside at the tumour edge, close to cancer-associated fibroblasts. Hence, stem cell properties change in time depending on the cell location. Furthermore, although chemotherapy enriches for cells with a CSC phenotype, in this context functional stem cell properties are also fully defined by the microenvironment. To conclude, we identified osteopontin as a key cancer-associated fibroblast-produced factor that drives in situ clonogenicity in colon cancer.

Colon cancer stem cells (CSCs) are closely related to tumorigenesis and treatment response, and LGR5 is currently the most robust and reliable CSC marker in colorectal cancer (CRC). However, LGR5 expression in CRC tumor budding (TB) is not well understood. We examined the clinicopathological and prognostic significance of LGR5 in CRC TB. LGR5 expression was evaluated by RNAscope, a newly developed RNA in situ hybridization technique, using a tissue microarray consisting of 55 patient samples of TB in colon adenocarcinoma (CA) selected from the medical archives at our hospital. Patients were stratified into negative and positive LGR5 expression groups. Inflammatory cell infiltration was weaker and histological grade was lower in the LGR5-positive group compared with the LGR5-negative group (P = 0.0407 and P = 0.0436, respectively). There was a significant difference in OS between the LGR5-positive group and LGR5-negative group (log-rank test, P = 0.0088). Cox proportional hazards models revealed that the LGR5-positive group (Overall survival (OS) = 0.37, 95% CI: 0.17–0.79, P = 0.0101) had better OS. LGR5 expression may be affected by inflammatory cell infiltration in the budding area of CA and is an important potential marker of prognosis.