Subtype-specific Expression Patterns Research Articles

Long Intergenic Non-Coding RNAs of Human Chromosome 18: Focus on Cancers.

Malignant neoplasms are characterized by high molecular heterogeneity due to multilevel deregulation of gene expression and cellular functions. It is known that non-coding RNAs, including long intergenic non-coding RNAs (lincRNAs), can play significant roles in cancer biology. The current review focuses on a systematical analysis of genomic, transcriptomic, epigenomic, interactomic, and literature data on 65 lincRNAs of human chromosome 18 in the context of pan-cancer studies. The entire group of lincRNAs can be conditionally divided into 4 subgroups depending on experimental evidence on direct or indirect involvement in cancers and the biological associations with cancers, which we found during the data-mining process: the most studied (5 lincRNAs), moderately or poorly studied (11 lincRNAs), and understudied (31 lincRNAs). For the remaining 18 lincRNAs, data for analysis were fragmentary or missing. Among the key findings were the following: Of the lincRNAs of human chromosome 18, 40% have tissue-specific expression patterns, 22% of lincRNAs are known to have gene fusions, 40% of lincRNAs are prone to gene amplifications and/or deletions in cancers at a frequency greater than 3%, and 23% of lincRNAs are differentially expressed across cancer types, whereas 7% have subtype-specific expression patterns. LincRNAs' interactomes consist of 'master' microRNAs and 47 proteins (including cancer-associated proteins and microRNAs) that can interact with 3 or more lincRNAs. Functional enrichment analysis of a set of highly co-expressed genes retrieved for 17 lincRNAs in different cancer types indicated the potential associations of these lincRNAs with cellular signaling pathways. Six lincRNAs encoded small open-reading frame (smORF) proteins with emerging roles in cancers, and microRNAs as well as proteins with known functions in molecular carcinogenesis can bind to coding regions of smORFs. We identified seven transcriptomic signatures with potential prognostic value, consisting of two to seven different lincRNAs only. Taken together, the literature, biomedical, and molecular biology data analyzed indicated that only five of all lincRNAs of human chromosome 18 are cancer-associated, while eleven other lincRNAs have the tendency to be associated with cancers.

Single-cell transcriptomics reveals subtype-specific molecular profiles in Nrf2-deficient macrophages from murine atherosclerotic aortas.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator of antioxidant and anti-inflammatory response in all cell types. It also activates the transcription of genes important for macrophage function. Nrf2 activity declines with age and has been closely linked to atherosclerosis, but its specific role in this vascular pathology is not clear. Atherosclerotic plaques contain several macrophage subsets with distinct, yet not completely understood, functions in the lesion development. The aim of this study was to analyze the transcriptome of diverse Nrf2-deficient macrophage subpopulations from murine atherosclerotic aortas. Mice with transcriptionally inactive Nrf2 in Cdh5-expressing cells (Nrf2 Cdh5tKO) were used in the experiments. These mice lack transcriptional Nrf2 activity in endothelial cells, but also in a proportion of leukocytes. We confirmed that the bone marrow-derived and tissue-resident macrophages isolated from Nrf2 Cdh5tKO mice exhibit a significant decline in Nrf2 activity. Atherosclerosis was induced in Nrf2 Cdh5tKO and appropriate control mice via adeno-associated viral vector (AAV)-mediated overexpression of murine proprotein convertase subtilisin/kexin type 9 (Pcsk9) in the liver and high-fat diet feeding. After 21 weeks, live aortic cells were sorted on FACS and single-cell RNA sequencing (scRNA-seq) was performed. Unsupervised clustering singled out 13 distinct aortic cell types. Among macrophages, 9 subclusters were identified. Differential gene expression analysis revealed cell subtype-specific expression patterns. A subset of inflammatory macrophages from atherosclerotic Nrf2 Cdh5tKO mice demonstrated downregulation of DNA replication genes (e.g. Mcm7, Lig1, Pola1) concomitant with upregulation of DNA damage sensor Atr gene. Atherosclerotic Nrf2 Cdh5tKO Lyve1+ resident macrophages showed strong upregulation of IFN-stimulated genes, as well as changes in the expression of death pathways-associated genes (Slc40a1, Bcl2a1). Furthermore, we observed subtype-specific expression of core ferroptosis genes (e.g. Cp, Hells, Slc40a1) in inflammatory versus tissue resident macrophages. This observation suggested a link between ferroptosis and inflammatory microenvironment appearing at a very early stage of atherogenesis. Our findings indicate that Nrf2 deficiency in aortic macrophages leads to subtype-specific transcriptomic changes associated with inflammation, iron homeostasis, cell injury or death pathways. This may help understanding the role of aging-associated decline of Nrf2 activity and the function of specific macrophage subtypes in atherosclerotic lesion development.

A combinatorial cis-regulatory logic restricts color-sensing Rhodopsins to specific photoreceptor subsets in Drosophila.

Color vision in Drosophila melanogaster is based on the expression of five different color-sensing Rhodopsin proteins in distinct subtypes of photoreceptor neurons. Promoter regions of less than 300 base pairs are sufficient to reproduce the unique, photoreceptor subtype-specific rhodopsin expression patterns. The underlying cis-regulatory logic remains poorly understood, but it has been proposed that the rhodopsin promoters have a bipartite structure: the distal promoter region directs the highly restricted expression in a specific photoreceptor subtype, while the proximal core promoter region provides general activation in all photoreceptors. Here, we investigate whether the rhodopsin promoters exhibit a strict specialization of their distal (subtype specificity) and proximal (general activation) promoter regions, or if both promoter regions contribute to generating the photoreceptor subtype-specific expression pattern. To distinguish between these two models, we analyze the expression patterns of a set of hybrid promoters that combine the distal promoter region of one rhodopsin with the proximal core promoter region of another rhodopsin. We find that the function of the proximal core promoter regions extends beyond providing general activation: these regions play a previously underappreciated role in generating the non-overlapping expression patterns of the different rhodopsins. Therefore, cis-regulatory motifs in both the distal and the proximal core promoter regions recruit transcription factors that generate the unique rhodopsin patterns in a combinatorial manner. We compare this combinatorial regulatory logic to the regulatory logic of olfactory receptor genes and discuss potential implications for the evolution of rhodopsins.

Identification of LAG3+ T Cell Populations in the Tumor Microenvironment of Classical Hodgkin Lymphoma and B-Cell Non-Hodgkin Lymphoma

Identification of LAG3+ T Cell Populations in the Tumor Microenvironment of Classical Hodgkin Lymphoma and B-Cell Non-Hodgkin Lymphoma

Evolutionary conservation of opsin gene expression patterns in the compound eyes of darkling beetles.

Recent large-scale studies of opsin gene contents in representatives of the largest order of insects, the Coleoptera (beetles), revealed that the blue wavelength-sensitive (B) opsin subfamily is absent in this clade, while the ultraviolet- (UV) and long wavelength-sensitive (LW) opsin subfamilies are broadly conserved with gene duplications possibly reintroducing blue sensitivity in select subclades. Little is known yet, however, how opsin genes are expressed in the compound eyes of beetles. In a previous study, we analyzed opsin gene expression in the red flour beetle Tribolium castaneum, a member of the family of darkling beetles (Tenebrionidae), and found that a singleton LW opsin homolog is homogeneously expressed in all photoreceptors of the compound eye retina with a singleton UV opsin homolog being co-expressed in the R7 subtype photoreceptors. To probe for the evolutionary conservation of these expression patterns, we isolated complete opsin transcript sequences from three additional species in the subfamily Tenebrionidae (Tribolium confusum, Tenebrio molitor, Zophobas morio) and studied their expression via whole mount in situ hybridization in the pupal retina. These experiments revealed very similar, if not identical, photoreceptor subtype-specific expression patterns in all three species compared with T. castaneum. Documenting a deep conservation of photoreceptor subtype-specific opsin gene expression in this range of darkling beetles, our study provides a first point of reference for broader comparative studies of retinal organization in the Coleoptera.

LINC00261 Is Differentially Expressed in Pancreatic Cancer Subtypes and Regulates a Pro-Epithelial Cell Identity.

Pancreatic adenocarcinoma (PDAC) is one of the major causes of cancer-associated deaths worldwide, with a dismal prognosis that has not significantly changed over the last decades. Transcriptional analysis has provided valuable insights into pancreatic tumorigenesis. Specifically, pancreatic cancer subtypes were identified, characterized by specific mutations and gene expression changes associated with differences in patient survival. In addition to differentially regulated mRNAs, non-coding RNAs, including long non-coding RNAs (lncRNAs), were shown to have subtype-specific expression patterns. Hence, we aimed to characterize prognostic lncRNAs with deregulated expression in the squamous subtype of PDAC, which has the worst prognosis. Extensive in silico analyses followed by in vitro experiments identified long intergenic non-coding RNA 261 (LINC00261) as a downregulated lncRNA in the squamous subtype of PDAC, which is generally associated with transforming growth factor β (TGFβ) signaling in human cancer cells. Its genomic neighbor, the transcription factor forkhead box protein A2 (FOXA2), regulated LINC00261 expression by direct binding of the LINC00261 promoter. CRISPR-mediated knockdown and promoter knockout validated the importance of LINC00261 in TGFβ-mediated epithelial–mesenchymal transition (EMT) and established the epithelial marker E-cadherin, an important cell adhesion protein, as a downstream target of LINC00261. Consequently, depletion of LINC00261 enhanced motility and invasiveness of PANC-1 cells in vitro. Altogether, our data suggest that LINC00261 is an important tumor-suppressive lncRNA in PDAC that is involved in maintaining a pro-epithelial state associated with favorable disease outcome.

Clinicopathological features and prognostic value of SOX11 in childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia is marked by aberrant transcriptional features that alter cell differentiation, self-renewal, and proliferative features. We sought to identify the transcription factors exhibiting altered and subtype-specific expression patterns in B-ALL and report here that SOX11, a developmental and neuronal transcription factor, is aberrantly expressed in the ETV6-RUNX1 and TCF3-PBX1 subtypes of acute B-cell leukemias. We show that a high expression of SOX11 leads to alterations of gene expression that are typically associated with cell adhesion, migration, and differentiation. A high expression is associated with DNA hypomethylation at the SOX11 locus and a favorable outcome. The results indicate that SOX11 expression marks a group of patients with good outcomes and thereby prompts further study of its use as a biomarker.

Distinct esophageal adenocarcinoma molecular subtype has subtype-specific gene expression and mutation patterns

BackgroundEsophageal carcinoma (EC), consists of two histological types, esophageal squamous carcinoma (ESCC) and esophageal adenocarcinoma (EAC). EAC accounted for 10% of EC for centuries; however, the prevalence of EAC has alarmingly risen 6 times and increased to about 50% of EC in recent 30 years in the western countries, while treatment options for EAC patients are still limited. Stratification of molecular subtypes by gene expression profiling methods had offered opportunities for targeted therapies. However, the molecular subtype in EAC has not been defined. Hence, Identification of EAC molecular subtypes is needed and will provide important insights for future new therapies.ResultsWe performed meta-analysis of gene expression profiling data on three independent EAC cohorts and showed that there are two common molecular subtypes in EAC. Each of the two EAC molecular subtypes has subtype specific expression patterns and mutation signatures. Genes which were over-expressed in subtype I EACs rather than subtype II EAC cases, were enriched in biological processes including epithelial cell differentiation, keratinocyte differentiation, and KEGG pathways including basal cell carcinoma. TP53 and CDKN2A are significantly mutated in both EAC subtypes. 24 genes including SMAD4 were found to be only significantly mutated in subtype I EAC cases, while 30 genes including ARID1A are only significantly mutated in subtype II EACs.ConclusionTwo EAC molecular subtypes were defined and validated. This finding may offer new opportunities for targeted therapies.

Duplicated membrane estrogen receptors in the European sea bass (Dicentrarchus labrax): Phylogeny, expression and regulation throughout the reproductive cycle

The numerous estrogen functions reported across vertebrates have been classically explained by their binding to specific transcription factors, the nuclear estrogen receptors (ERs). Rapid non-genomic estrogenic responses have also been recently identified in vertebrates including fish, which can be mediated by membrane receptors such as the G protein-coupled estrogen receptor (Gper). In this study, two genes for Gper, namely gpera and gperb, were identified in the genome of a teleost fish, the European sea bass. Phylogenetic analysis indicated they were most likely retained after the 3R teleost-specific whole genome duplication and raises questions about their function in male and female sea bass. Gpera expression was mainly restricted to brain and pituitary in both sexes while gperb had a widespread tissue distribution with higher expression levels in gill filaments, kidney and head kidney. Both receptors were detected in the hypothalamus and pituitary of both sexes and significant changes in gpers expression were observed throughout the annual reproductive season. In female pituitaries, gpera showed an overall increase in expression throughout the reproductive season while gperb levels remained constant. In the hypothalamus, gpera had a higher expression during vitellogenesis and decreased in fish entering the ovary maturation and ovulation stage, while gperb expression increased at the final atresia stage. In males, gpers expression was constant in the hypothalamus and pituitary throughout the reproductive cycle apart from the mid- to late testicular development stage transition when a significant up-regulation of gpera occurred in the pituitary. The differential sex, seasonal and subtype-specific expression patterns detected for the two novel gper genes in sea bass suggests they may have acquired different and/or complementary roles in mediating estrogens actions in fish, namely on the neuroendocrine control of reproduction2Protein and gene nomenclature followed that recommended by genenames.org and used for fish at http://zfin.org/; in this abbreviation list for each case protein abbreviation is presented first followed by the corresponding gene abbreviation..

Abstract 3407: Gene expression subtypes of high grade serous ovarian cancer in African American women

Abstract Ovarian cancer accounts for 5% of cancer deaths and is the fifth leading cause of cancer death in women in the United States. While incidence is higher in European American (EA) than African American (AA) women, five-year survival is worse for AA women (36%) than EA women (44%). Access to appropriate surgery and treatment is a major contributor but does not completely explain this disparity. The Cancer Genome Atlas (TCGA) identified four gene expression-based subtypes of the most common and lethal histotype, high grade serous carcinoma (HGSC): mesenchymal, proliferative, differentiated, and immunoreactive. We sought to characterize similarities and differences in gene expression-based subtypes arising in AA and EA women to determine whether there are underlying biologic features that may influence survival. We performed two distinct analyses, first using TCGA data and second using cases from the population-based African American Cancer Epidemiology Study (AACES). For both we summarized differential expression patterns for each subtype with moderated t statistic vectors for >10,000 genes using Significance Analysis of Microarrays. We calculated Pearson's correlations of these vectors to determine concordance of expression patterns between subtypes across EA and AA women. In TCGA, we observed correlations of subtype-specific expression patterns between the 24 AA and 475 EA tumors of 0.52-0.60 for each of the four subtypes. Thus, while analogous subtypes can be identified in AA and EA women, the magnitude of these correlations suggests that there are potential differences in gene expression patterns between AA and EA tumors that are assigned to the same subtype. We generated additional data from 58 AACES HGSC cases using the Affymetrix Human Transcriptome Array 2.0. Instead of assigning these tumors to previously-defined subtypes, we clustered samples to identify four subtypes de novo. We observed concordance with two of the TCGA subtypes; correlations for the mesenchymal-like and proliferative-like subtypes were 0.56-0.65. The mesenchymal-like subtype was more common in these AA women than in the TCGA EA women (33% versus 25%), and the proliferative-like subtype was marginally less common (14% versus 19%). Concordance for the differentiated-like subtype was considerably lower, at 0.21, and this subtype was less common in AA than EA women (19% versus 34%). Another subtype comprising 34% of the AA samples was only weakly correlated (-0.21-0.10) with any of the TCGA subtypes, suggesting that it is a novel subtype. The limited data available on HGSC in AA women suggest that at least two subtypes are comparable to those in EA women but differ in prevalence, and that there may be a novel subtype in AA women that does not strongly correspond to those described in EA women. Citation Format: Jennifer A. Doherty, Casey S. Greene, James E. Rudd, Laura J. Tafe, Anthony J. Alberg, Elisa V. Bandera, Jill Barnholtz-Sloan, Melissa Bondy, Michele L. Cote, Ellen Funkhouser, Patricia G. Moorman, Edward S. Peters, Ann G. Schwartz, Paul Terry, Rex Bentley, Andrew Berchuck, Jeffrey R. Marks, Joellen M. Schildkraut. Gene expression subtypes of high grade serous ovarian cancer in African American women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3407.

A novel subtype classification and risk of breast cancer by histone modification profiling.

Breast cancer has been classified into several intrinsic molecular subtypes on the basis of genetic and epigenetic factors. However, knowledge about histone modifications that contribute to the classification and development of biologically distinct breast cancer subtypes remains limited. Here we compared the genome-wide binding patterns of H3K4me3 and H3K27me3 between human mammary epithelial cells and three breast cancer cell lines representing the luminal, HER2, and basal subtypes. We characterized thousands of unique binding events as well as bivalent chromatin signatures unique to each cancer subtype, which were involved in different epigenetic regulation programs and signaling pathways in breast cancer progression. Genes linked to the unique histone mark features exhibited subtype-specific expression patterns, both in cancer cell lines and primary tumors, some of which were confirmed by qPCR in our primary cancer samples. Finally, histone mark-based gene classifiers were significantly correlated with relapse-free survival outcomes in patients. In summary, we have provided a valuable resource for the identification of novel biomarkers of subtype classification and clinical prognosis evaluation in breast cancers.

Glycan-related gene expression signatures in breast cancer subtypes; relation to survival

Alterations in glycan structures are early signs of malignancy and have recently been proposed to be in part a driving force behind malignant transformation. Here, we explore whether differences in expression of genes related to the process of glycosylation exist between breast carcinoma subtypes – and look for their association to clinical parameters.Five expression datasets of 454 invasive breast carcinomas, 31 ductal carcinomas in situ (DCIS), and 79 non-malignant breast tissue samples were analysed. Results were validated in 1960 breast carcinomas. 419 genes encoding glycosylation-related proteins were selected.The DCIS samples appeared expression-wise similar to carcinomas, showing altered gene expression related to glycosaminoglycans (GAGs) and N-glycans when compared to non-malignant samples. In-situ lesions with different aggressiveness potentials demonstrated changes in glycosaminoglycan sulfation and adhesion proteins. Subtype-specific expression patterns revealed down-regulation of genes encoding glycan-binding proteins in the luminal A and B subtypes. Clustering basal-like samples using a consensus list of genes differentially expressed across discovery datasets produced two clusters with significantly differing prognosis in the validation dataset. Finally, our analyses suggest that glycolipids may play an important role in carcinogenesis of breast tumors – as demonstrated by association of B3GNT5 and UGCG genes to patient survival.In conclusion, most glycan-specific changes occur early in the carcinogenic process. We have identified glycan-related alterations specific to breast cancer subtypes including a prognostic signature for two basal-like subgroups. Future research in this area may potentially lead to markers for better prognostication and treatment stratification of breast cancer patients.

Expression of neurotrophins and their receptors in adult sarcomas.

10562 Background: Neurotrophins (NT) influence proliferation, survival, death, and differentiation by binding Tropomyosin-related kinase (Trk) receptors and p75NTR(CD271). While the three Trk-receptors TrkA, TrkB, and TrkC display specific binding to their ligands, i.e. TrkA to NGF (nerve growth factor), TrkB to BDNF (brain-derived neurotrophic factor), NT4/5 (neurotrophin 4/5) and TrkC to NT3 (neurotrophin 3), CD271 shows low affinity to all neurotrophins. Notably, CD271 has been suggested as marker for the prospective isolation of bone marrow mesenchymal stromal cells (MSC), and rhabdomyosarcomas have previously been found to express neurotrophins and NT receptors. Methods: RNA was isolated from sarcoma cell lines and freshly obtained sarcoma (src) cells from patients after surgical resection. RNA was reverse transcribed and cDNA products underwent PCR analysis with specific primers. Furthermore, cell lines from the same primary tissues were established and treated with doxorubicin. Effects of K252a (Trk receptor inhibitor), Pep5 (CD271-Inhibitor), and neurotrophins on sarcoma cell proliferation and survival were analyzed. Results: An array of freshly obtained clinical src samples (n=10 each subtype) was screened for expression of NT3, NT4/5, BDNF, NGF as well as TrkA, TrkB, TrkC, and CD271. Interestingly, src subtype-specific expression patterns were observed. In the established cell lines, K252a increased doxorubicin-induced tumor cell death, while CD271-Inhibitor Pep5 enhanced proliferation in doxorubicin treated cells, especially under serum free conditions and when combined with BDNF and NGF. Conclusions: NT and NT receptors are expressed on all adult src samples screened in this study. Chemotherapy resistant liposrc as well as myxofibrosrc samples displayed the strongest expression of NT3 as well as TrkB, TrkC, and CD271. The concomitant expression of NT and their receptors suggests autocrine self-stimulation, which may propagate survival, proliferation, and treatment resistance. NT receptor tyrosine kinases may represent an interesting therapeutic target in adult src.

Molecular subtypes of breast cancer are associated with characteristic DNA methylation patterns.

IntroductionFive different molecular subtypes of breast cancer have been identified through gene expression profiling. Each subtype has a characteristic expression pattern suggested to partly depend on cellular origin. We aimed to investigate whether the molecular subtypes also display distinct methylation profiles.MethodsWe analysed methylation status of 807 cancer-related genes in 189 fresh frozen primary breast tumours and four normal breast tissue samples using an array-based methylation assay.ResultsUnsupervised analysis revealed three groups of breast cancer with characteristic methylation patterns. The three groups were associated with the luminal A, luminal B and basal-like molecular subtypes of breast cancer, respectively, whereas cancers of the HER2-enriched and normal-like subtypes were distributed among the three groups. The methylation frequencies were significantly different between subtypes, with luminal B and basal-like tumours being most and least frequently methylated, respectively. Moreover, targets of the polycomb repressor complex in breast cancer and embryonic stem cells were more methylated in luminal B tumours than in other tumours. BRCA2-mutated tumours had a particularly high degree of methylation. Finally, by utilizing gene expression data, we observed that a large fraction of genes reported as having subtype-specific expression patterns might be regulated through methylation.ConclusionsWe have found that breast cancers of the basal-like, luminal A and luminal B molecular subtypes harbour specific methylation profiles. Our results suggest that methylation may play an important role in the development of breast cancers.

Islet1 and Islet2 have equivalent abilities to promote motoneuron formation and to specify motoneuron subtype identity

The expression of LIM homeobox genes islet1 and islet2 is tightly regulated during development of zebrafish primary motoneurons. All primary motoneurons express islet1 around the time they exit the cell cycle. By the time primary motoneurons undergo axogenesis, specific subtypes express islet1, whereas other subtypes express islet2, suggesting that these two genes have different functions. Here, we show that Islet1 is required for formation of zebrafish primary motoneurons; in the absence of Islet1, primary motoneurons are missing and there is an apparent increase in some types of ventral interneurons. We also provide evidence that Islet2 can substitute for Islet1 during primary motoneuron formation. Surprisingly, our results demonstrate that despite the motoneuron subtype-specific expression patterns of Islet1 and Islet2, the differences between the Islet1 and Islet2 proteins are not important for specification of the different primary motoneuron subtypes. Thus, primary motoneuron subtypes are likely to be specified by factors that act in parallel to or upstream of islet1 and islet2.

Molecular cloning of canine monoamine oxidase subtypes A (MAOA) and B (MAOB) cDNAs and their expression in the brain.

The role of monoamine oxidase has been shown to be related to some behavioral changes including aggression and cognitive dysfunction. In order to demonstrate the basic expression patterns of monoamine oxidase in the canine brain, we determined the full-length nucleotide sequences of cDNA for canine monoamine oxidase type A (MAOA) and type B (MAOB) genes that were isolated from the canine brain cDNA library. Oligonucleotide primers for PCR were constructed based on the conserved sequences reported thus far for other mammalian species. The nucleotide sequences had open reading frames of 1584 and 1563 bp for MAOA and MAOB, respectively. Both of these genes showed relatively high homology with other species in both nucleotide (> 81%) and deduced amino acid (> 85%) sequences. In Northern blot analyses MAOA mRNA was expressed broadly in various parts of the canine brain, whereas MAOB mRNA was found only in specific brain regions, such as the hypothalamus, hippocampus, brain stem and olfactory bulb. These results suggest that MAOA and MAOB mRNAs have subtype-specific expression patterns in the canine brain.

Subtype-specific expression patterns of inositol 1,4,5-trisphosphate receptors in rat airway epithelial cells.

We investigated the immunohistochemical localization of inositol 1,4,5-trisphosphate receptor (IP3R) Types 1, 2, and 3 in rat airway epithelium using the monoclonal antibodies KM1112, KM1083, and KM1082 specific for each type of IP3R. The epithelium from trachea to distal intrapulmonary airways (bronchioles) showed positive immunoreactivity for all types of IP3R. However, cell type as well as subcellular site immunoreactivity for each type of IP3R varied. IP3R Type 1 was found only in the apical thin cytoplasmic area of ciliated cells throughout all airway levels. IP3R Type 2 was exclusively localized to the entire cytoplasm of ciliated cells from the trachea to bronchioles. IP3R Type 3 was expressed mainly in the supranuclear cytoplasm not only of ciliated cells at all airway levels but also in Clara cells of the bronchiolar epithelium. Double fluorescent staining using combinations of KM1083 and Wisteria floribunda lectin or anti-rat 10-KD Clara cell-specific protein antibody confirmed that the IP3R Type 2-positive cells were neither seromucous cells nor Clara cells. These results indicate that the expression of three types of IP3Rs in different cell types and subcellular sites may reflect diverse physiological functions of IP3Rs within airway epithelial cells. The double staining studies suggested that the anti-IP3R Type 2 monoclonal antibody KM1083 would be a specific cell marker for ciliated cells of the airway epithelium.