Characterization of Primary Cilia in Normal Fallopian Tube Epithelium and Serous Tubal Intraepithelial Carcinoma.
ABSTRACT: OBJECTIVES:The aim of this study was to investigate the distribution of primary cilia on secretory cells in normal fallopian tube (FT) and serous tubal intraepithelial carcinoma (STIC). METHODS:Fallopian tube tissue samples were obtained from 4 females undergoing prophylactic hysterectomies and 6 patients diagnosed with STIC. A mogp-TAg transgenic mouse STIC sample was also compared with a wild-type mouse FT sample. Serous tubal intraepithelial carcinoma was identified by hematoxylin and eosin staining and confirmed by positive Ki-67 and p53 immunohistochemical staining of tissue sections. We assessed the relative distribution of primary cilia on secretory cells and motile cilia on multiple ciliated cells by immunofluorescence and immunohistochemical staining. Ciliary function was assessed by immunofluorescence staining of specific ciliary marker proteins and responsiveness to Sonic Hedgehog signaling. RESULTS:Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of human FT where they may appear to mediate ciliary-mediated Sonic Hedgehog signaling. A statistically significant reduction in the number of primary cilia on secretory cells was observed in human STIC samples compared with normal controls (P < 0.0002, Student t test), supported by similar findings in a mouse STIC sample. Immunohistochemical staining for dynein axonemal heavy chain 5 discriminated multiple motile cilia from primary cilia in human FT. CONCLUSIONS:Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of the human FT but are significantly reduced in both human and mouse STIC samples. Immunohistochemical staining for ciliary proteins may have clinical utility for early detection of STIC.
Project description:There is compelling evidence to suggest that serous tubal intraepithelial carcinoma (STIC) is the likely primary site for the development of many pelvic high-grade serous carcinomas (HGSCs). Identifying molecules that are upregulated in STIC is important not only to provide biomarkers to assist in the diagnosis of STIC but also to elucidate our understanding of the pathogenesis of HGSC. In this study, we performed RNA sequencing to compare transcriptomes between HGSC and normal fallopian tube epithelium (FTE), and we identified LAMC1 encoding laminin ?1 as one of the preferentially upregulated genes associated with HGSC. Reverse transcription polymerase chain reaction further validated LAMC1 upregulation in HGSC as compared with normal FTE. Immunohistochemical analysis was performed on 32 cases of concurrent HGSC and STIC. The latter was diagnosed on the basis of morphology, TP53 mutations, and p53 and Ki-67 immunohistochemical patterns. Laminin ?1 immunostaining intensity was found to be significantly higher in STIC and HGSC compared with adjacent FTE in all cases (P<0.001). In normal FTE, laminin ?1 immunoreactivity was predominantly localized in the basement membrane or on the apical surface of ciliated cells, whereas in STIC and HGSC cells, laminin ?1 staining was diffuse and intense throughout the cytoplasm. More importantly, strong laminin ?1 staining was detected in all 13 STICs, which lacked p53 immunoreactivity because of null mutations. These findings suggest that the overexpression of laminin ?1 immunoreactivity and alteration of its staining pattern in STICs can serve as a useful tissue biomarker, especially for those STICs that are negative for p53 and have a low Ki-67 labeling index.
Project description:Cilia and flagella are highly conserved organelles that have diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia and flagella often result in primary ciliary dyskinesia. However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a new gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in primary ciliary dyskinesia patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.
Project description:The present review focuses on the current status of molecular pathology in high-grade serous cancer (HGSC) and preneoplastic conditions. This article reviews the English-language literature on HGSC, precursor, fallopian tubal epithelium, secretory cells, ciliated cells, secretory cell expansion, secretory cell outgrowth (SCOUT), p53 signature, serous tubal intraepithelial carcinoma (STIC), DNA damage and immunohistochemistry in an effort to identify the precursor-carcinoma sequence in HGSC. The majority of HGSC originates from the fimbriated end of the fallopian tube secretory epithelial cells, while the small part of this disease may develop from ovarian cortical inclusion cyst (CIC). A series of morphological changes from normal fallopian epithelium to preneoplastic to neoplastic lesions were concomitant with the multistep accumulation of molecular and genetic alterations. Recent studies provide a stepwise progression of fallopian tubal epithelium to precursor lesions to carcinoma, with the aid of a 'secretory cell-SCE-SCOUT-p53 signature-STIC-HGSC sequence' model. Immunohistochemical markers, including p53, STMN1, EZH2, CCNE1, Ki67 and γ-H2AX, were gradually increased during the SCOUT-p53 signature-STIC-HGSC sequence. Conversely, PAX2 expression was decreased during the early phase of SCOUT development. Potential genes and proteins are involved in the evolutionary trajectory of the precursor-cancer lineage model. In the present review we examined detailed aspects of the molecular changes involved in malignant transformation from fallopian tube epithelium to HGSC. A precursor condition originating in 'field cancerization' may gain a growth advantage, leading to HGSC.
Project description:While cilia are recognized as important signaling organelles, the extent of ciliary functions remains unknown because of difficulties in cataloguing proteins from mammalian primary cilia. We present a method that readily captures rapid snapshots of the ciliary proteome by selectively biotinylating ciliary proteins using a cilia-targeted proximity labeling enzyme (cilia-APEX). Besides identifying known ciliary proteins, cilia-APEX uncovered several ciliary signaling molecules. The kinases PKA, AMPK, and LKB1 were validated as bona fide ciliary proteins and PKA was found to regulate Hedgehog signaling in primary cilia. Furthermore, proteomics profiling of Ift27/Bbs19 mutant cilia correctly detected BBSome accumulation inside Ift27(-/-) cilia and revealed that ?-arrestin 2 and the viral receptor CAR are candidate cargoes of the BBSome. This work demonstrates that proximity labeling can be applied to proteomics of non-membrane-enclosed organelles and suggests that proteomics profiling of cilia will enable a rapid and powerful characterization of ciliopathies.
Project description:Primary cilia protrude from the cell surface of many cell types in the human body and function as cellular antennae via ciliary membrane localized receptors. Neurons and glial cells in the brain possess primary cilia, and the malfunction of primary cilia may contribute to neurological deficits present in many cilia-associated disorders. Several rhodopsin family G-protein coupled receptors (GPCRs) are specifically localized to a subset of neuronal primary cilia. However, whether other family GPCRs target to neuronal cilia and whether glial primary cilia harbor any GPCRs are not known. We conducted a screening of GPCRs to determine their ability to target to primary cilia, and identified a secretin family member, Vasoactive Intestinal Receptor 2 (VPAC2), as a novel ciliary GPCR. Here, we show that endogenous VPAC2 targets to primary cilia in various brain regions, including the suprachiasmatic nuclei and the thalamus. Surprisingly, VPAC2 not only localizes to neuronal cilia but also to glial cilia. In addition, we show that VPAC2's C-terminus is both necessary and sufficient for its ciliary targeting and we define a novel ciliary targeting signal: the tetrapeptide RDYR motif in the C-terminus of VPAC2. Furthermore, we demonstrate that VPAC2 ciliary targeting is dependent on Tubby, the BBSome (a complex of Bardet-Biedl syndrome proteins) and the BBSome targeting factor, Arl6.
Project description:<h4>Unlabelled</h4><h4>Background</h4>Cilia are essential organelles in multiple organ systems, including the kidney where they serve as important regulators of renal homeostasis. Renal nephron cilia emanate from the apical membrane of epithelia, extending into the lumen where they function in flow-sensing and ligand-dependent signaling cascades. Ciliary dysfunction underlies renal cyst formation that is in part caused by deregulation of planar cell polarity and canonical Wnt signaling. Renal cancer pathologies occur sporadically or in heritable syndromes caused by germline mutations in tumor suppressor genes including VHL. Importantly, Von Hippel-Lindau (VHL) patients frequently develop complex renal cysts that can be considered a premalignant stage. One of the well-characterized molecular functions of VHL is its requirement for the maintenance of cilia. In this study, tissue from 110 renal cancer patients who underwent nephrectomy was analyzed to determine if lower ciliary frequency is a common hallmark of renal tumorigenesis by comparing cilia frequencies in both tumor and adjacent parenchymal tissue biopsies from the same kidney.<h4>Methods</h4>We stained sections of human renal material using markers for cilia. Preliminary staining was performed using an immunofluorescent approach and a combination of acetylated-?-tubulin and pericentrin antibodies and DAPI. After validation of an alternative, higher throughput approach using acetylated-?-tubulin immunohistochemistry, we continued to manually quantify cilia in all tissues. Nuclei were separately counted in an automated fashion in order to determine ciliary frequencies. Similar staining and scoring for Ki67 positive cells was performed to exclude that proliferation obscures cilia formation potential.<h4>Results</h4>Samples from renal cell carcinoma patients deposited in our hospital tissue bank were previously used to compose a tissue microarray containing three cores of both tumor and parenchymal tissue per patient. Cilia frequencies in a total of eighty-nine clear cell, eight papillary, five chromophobe renal cell carcinomas, two sarcomatoid renal tumors and six oncocytomas were determined. A marked decrease of primary cilia across renal cell carcinoma subtypes was observed compared to adjacent nontumorigenic tissue.<h4>Conclusions</h4>Our study shows that cilia are predominantly lost in renal cell carcinomas compared to tissue of the tumor parenchyma. These results suggest that ciliary loss is common in renal tumorigenesis, possibly participating in the sequence of cellular events leading to malignant tumor development. Future therapies aimed at restoring or circumventing cilia signaling might therefore aid in current treatment efficacy.
Project description:The aim of this study was to compare the transcriptome of the different regions of the oviduct between pregnant and cyclic heifers. After synchronizing crossbred beef heifers, those in standing oestrus (=Day 0) were randomly assigned to cyclic (non bred, n=6), or pregnant (artificially inseminated, n=11) groups. They were slaughtered on Day 3 and both oviducts from each animal were isolated and cut in half to separate ampulla and isthmus. Each portion was flushed to confirm the presence of an oocyte/embryo and was then opened longitudinally and scraped to obtain epithelial cells which were snap-frozen. Oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum. Microarray analysis of oviductal cells revealed that proximity to the corpus luteum did not affect the transcriptome of the isthmus, irrespective of pregnancy status. However, 2287 genes were differentially expressed (P<0.01) between the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum. Gene ontology revealed that the main biological processes overrepresented in the isthmus were synthesis of nitrogen, lipids, nucleotides, steroids and cholesterol as well as vesicle-mediated transport, cell cycle, apoptosis, endocytosis and exocytosis, whereas cell motion, motility and migration, DNA repair, calcium ion homeostasis, carbohydrate biosynthesis and regulation of cilium movement and beat frequency were overrepresented in the ampulla. In conclusion, large differences in gene expression were observed between the isthmus and ampulla that reflect morphological and functional characteristics of each segment. Fifteen samples including 5 pregnant contralateral isthmus heifers, 5 cyclic contralateral isthmus heifers, and 5 pregnant ipsilateral ampulla heifers. Also, ten samples from GSE74593 were reanalyzed with these samples. The full set of processed data are linked at the bottom of the Series record.
Project description:Reproductive success depends on a functional oviduct for gamete storage, maturation, fertilization, and early embryonic development. The ovarian-derived steroids estrogen and progesterone are key regulators of oviductal function. The objective of this study was to investigate luteal and follicular phase-specific oviductal epithelial cell function by using microarray-based transcriptional profiling, to increase our understanding of mRNAs regulating epithelial cell processes, and to identify novel genes and biochemical pathways that may be found to affect fertility in the future.Six normally cycling Angus heifers were assigned to either luteal phase (LP, n?=?3) or follicular phase (FP, n?=?3) treatment groups. Heifers in the LP group were killed between day 11 and 12 after estrus. Heifers in the FP group were treated with 25 mg PGF2? (Lutalyse, Pfizer, NY) at 8 pm on day 6 after estrus and killed 36 h later. Transcriptional profiling by microarray and confirmation of selected mRNAs by real-time RT-PCR analyses was performed using total RNA from epithelial cells isolated from sections of the ampulla and isthmus collected from LP and FP treatment groups. Differentially expressed genes were subjected to gene ontology classification and bioinformatic pathway analyses.Statistical one-way ANOVA using Benjamini-hochberg multiple testing correction for false discovery rate (FDR) and pairwise comparison of epithelial cells in the ampulla of FP versus LP groups revealed 972 and 597 transcripts up- and down-regulated, respectively (P?<?0.05). Within epithelial cells of the isthmus in FP versus LP groups, 946 and 817 transcripts were up- and down-regulated, respectively (P?<?0.05). Up-regulated genes from both ampulla and isthmus were found to be largely involved in cholesterol biosynthesis and cell cycle pathways, while down-regulated genes were found in numerous inflammatory response pathways.Microarray-based transcriptional profiling revealed phase of the cycle-dependent changes in the expression of mRNA within the epithelium of the oviducts' ampulla and isthmus.
Project description:Primary cilia are essential sensory and signaling organelles present on nearly every mammalian cell type. Defects in primary cilia underlie a class of human diseases collectively termed ciliopathies. Primary cilia are restricted subcellular compartments, and specialized mechanisms coordinate the localization of proteins to cilia. Moreover, trafficking of proteins into and out of cilia is required for proper ciliary function, and this process is disrupted in ciliopathies. The somatostatin receptor subtype 3 (Sstr3) is selectively targeted to primary cilia on neurons in the mammalian brain and is implicated in learning and memory. Here, we show that Sstr3 localization to cilia is dynamic and decreases in response to somatostatin treatment. We further show that somatostatin treatment stimulates ?-arrestin recruitment into Sstr3-positive cilia and this recruitment can be blocked by mutations in Sstr3 that impact agonist binding or phosphorylation. Importantly, somatostatin treatment fails to decrease Sstr3 ciliary localization in neurons lacking ?-arrestin 2. Together, our results implicate ?-arrestin in the modulation of Sstr3 ciliary localization and further suggest a role for ?-arrestin in the mediation of Sstr3 ciliary signaling.