Targeting HTR2B suppresses non-functioning pituitary adenoma growth and sensitizes cabergoline treatment via inhibiting Gαq/PLC/PKCγ/STAT3 axis.

BACKGROUND: Managing non-functioning pituitary adenomas (NFPAs) is difficult due to limited drug treatments. Cabergoline's (CAB) effectiveness for NFPAs is debated. This study explores the role of HTR2B in NFPAs and its therapeutic potential. METHODS: We conducted screening of bulk RNA-sequencing data to analyze HTR2B expression levels in NFPA samples. In vitro and in vivo experiments were performed to evaluate the effects of HTR2B modulation on tumor growth and cell cycle regulation. Mechanistic insights into the HTR2B-mediated signaling pathway were elucidated using pharmacological inhibitors and molecular interaction assays. RESULTS: Elevated HTR2B expression was detected in NFPA samples, which was associated with increased tumor survival. Inhibition of HTR2B activity resulted in the suppression of tumor growth through modulation of the G2M cell cycle. The inhibition of HTR2B with PRX-08066 was found to block STAT3 phosphorylation and nuclear translocation by interfering with the Gαq/PLC/PKC pathway. A direct interaction between PKC-γ and STAT3 was critical for STAT3 activation. CAB was shown to activate pSTAT3 via HTR2B, reducing its therapeutic potential. However, the combination of an HTR2B antagonist with CAB significantly inhibited tumor cell proliferation in HTR2B-expressing pituitary tumor cell lines, a xenografted pituitary tumor model, and patient-derived samples. Analysis of patient-derived data indicated that a distinct molecular pattern characterized by upregulated HTR2B/PKC-γ and downregulated BTG2/GADD45A may benefit from combination treatment with CAB and PRX-08066. CONCLUSIONS: HTR2B is a potential therapeutic target for NFPAs, and its inhibition could improve CAB efficacy. A dual therapy approach may be beneficial for NFPA patients with high HTR2B expression.

Therapeutic potential of targeting Nrf2 by panobinostat in pituitary neuroendocrine tumors.

We aimed to identify the druggable cell-intrinsic vulnerabilities and target-based drug therapies for PitNETs using the high-throughput drug screening (HTS) and genomic sequencing methods. We examined 9 patient-derived PitNET primary cells in HTS. Based on the screening results, the potential target genes were analyzed with genomic sequencing from a total of 180 PitNETs. We identified and verified one of the most potentially effective drugs, which targeted the Histone deacetylases (HDACs) both in in vitro and in vivo PitNET models. Further RNA sequencing revealed underlying molecular mechanisms following treatment with the representative HDACs inhibitor, Panobinostat. The HTS generated a total of 20,736 single-agent dose responses which were enriched among multiple inhibitors for various oncogenic targets, including HDACs, PI3K, mTOR, and proteasome. Among these drugs, HDAC inhibitors (HDACIs) were, on average, the most potent drug class. Further studies using in vitro, in vivo, and isolated PitNET primary cell models validated HDACIs, especially Panobinostat, as a promising therapeutic agent. Transcriptional surveys revealed substantial alterations to the Nrf2 signaling following Panobinostat treatment. Moreover, Nrf2 is highly expressed in PitNETs. The combination of Panobinostat and Nrf2 inhibitor ML385 had a synergistic effect on PitNET suppression. The current study revealed a class of effective anti-PitNET drugs, HDACIs, based on the HTS and genomic sequencing. One of the representative compounds, Panobinostat, may be a potential drug for PitNET treatment via Nrf2-mediated redox modulation. Combination of Panobinostat and ML385 further enhance the effectiveness for PitNET treatment.

Single-cell transcriptomics reveal distinct immune-infiltrating phenotypes and macrophage-tumor interaction axes among different lineages of pituitary neuroendocrine tumors.

BACKGROUND: Pituitary neuroendocrine tumors (PitNETs) are common gland neoplasms demonstrating distinctive transcription factors. Although the role of immune cells in PitNETs has been widely recognized, the precise immunological environment and its control over tumor cells are poorly understood. METHODS: The heterogeneity, spatial distribution, and clinical significance of macrophages in PitNETs were analyzed using single-cell RNA sequencing (scRNA-seq), bulk RNA-seq, spatial transcriptomics, immunohistochemistry, and multiplexed quantitative immunofluorescence (QIF). Cell viability, cell apoptosis assays, and in vivo subcutaneous xenograft experiments have confirmed that INHBA-ACVR1B influences the process of tumor cell apoptosis. RESULTS: The present study evaluated scRNA-seq data from 23 PitNET samples categorized into 3 primary lineages. The objective was to explore the diversity of tumors and the composition of immune cells across these lineages. Analyzed data from scRNA-seq and 365 bulk RNA sequencing samples conducted in-house revealed the presence of three unique subtypes of tumor immune microenvironment (TIME) in PitNETs. These subtypes were characterized by varying levels of immune infiltration, ranging from low to intermediate to high. In addition, the NR5A1 lineage is primarily associated with the subtype characterized by limited infiltration of immune cells. Tumor-associated macrophages (TAMs) expressing CX3CR1+, C1Q+, and GPNMB+ showed enhanced contact with tumor cells expressing NR5A1 + , TBX19+, and POU1F1+, respectively. This emphasizes the distinct interaction axes between TAMs and tumor cells based on their lineage. Moreover, the connection between CX3CR1+ macrophages and tumor cells via INHBA-ACVR1B regulates tumor cell apoptosis. CONCLUSIONS: In summary, the different subtypes of TIME and the interaction between TAM and tumor cells offer valuable insights into the control of TIME that affects the development of PitNET. These findings can be utilized as prospective targets for therapeutic interventions.

Alternations of Blood Pressure Following Surgical or Drug Therapy for Prolactinomas.

Several subtypes of pituitary neuroendocrine tumors (PitNETs), such as acromegaly and Cushing's disease, can result in hypertension. However, whether prolactinoma is associated with this complication remains unknown. Moreover, the effect of treatment with surgery or drugs on blood pressure (BP) is unknown. Herein, a retrospective study reviewed 162 patients with prolactinoma who underwent transsphenoidal surgery between January 2005 and December 2022. BP measurements were performed 1 day before and 5 days after surgery. Accordingly, patients' medical characteristics were recorded. In addition, in situ rat and xenograft nude-mice prolactinoma models have been used to mimic prolactinoma. In vivo BP and serum prolactin (PRL) levels were measured after cabergoline (CAB) administration in both rats and mice. Our data suggest that surgery can effectively decrease BP in prolactinoma patients with or without hypertension. The BP-lowering effect was significantly associated with several variables, including age, sex, disease duration, tumor size, invasion, dopamine agonists (DAs)-resistance, recurrence, and preoperative PRL levels. Moreover, in situ and xenograft prolactinomas induced BP elevation, which was alleviated by CAB treatment without and with a statistical difference in rats and mice, respectively. Thus, surgery or CAB can decrease BP in prolactinoma, indicating that pre- and postoperative BP management becomes essential.

Fully endoscopic far-lateral supracerebellar infratentorial approach for trigeminal neuralgia: illustrative case reports.

BACKGROUND: Trigeminal neuralgia (TN) is a common cause of craniofacial pain. The retrosigmoid approach is usually used to treat TN, but no cases of endoscopic far-lateral supracerebellar infratentorial approach (EF-SCITA) were used to undergo operation for TN. CASE PRESENTATION: Two patients were presented with severe facial pain and preliminary diagnosis was TN. Preoperative magnetic resonance imaging revealed that a superior cerebellar artery (SCA) compressed the trigeminal nerve in case 1, and a tumor located in the petrous apex extending into the Meckel's cave compressed the trigeminal nerve in case 2. Operations were achieved through the EF-SCITA. The pain was totally relieved with no postsurgical complications in both cases. CONCLUSIONS: We present the first two case reports of EF-SCITA to relieve classical and secondary TN successfully. The EF-SCITA can be a promising approach for treating TN.

Upper tract urothelial carcinoma accompanied by hyperthermia: A case report.

Upper tract urothelial carcinoma (UTUC) is a relatively rare malignant neoplasm of the urinary system. Due to its highly aggressiveness, the tumor has already undergone invasive growth when most UTUC patients are diagnosed. In addition, the most common cause of fever in cancer patients is infection, and cancer patients with neoplastic fever are relatively rare. We reported a 58-year-old man with invasive high-grade UTUC accompanied by hyperthermia.

DRD2 expression based on 18F-fallypride PET/MR predicts the dopamine agonist resistance of prolactinomas: a pilot study.

PURPOSE: The dopamine agonists (DA) have been used widely to treat prolactinomas. However, it is difficult to predict whether the patient will be responsive to DA treatment. METHODS: We aimed to investigate whether the in vivo expression of DRD2 based on 18F-fallypride PET/MR could predict the therapeutic effect of DA on prolactinomas. Seven patients with prolactinomas completed 18F-fallypride PET/MR. Among them, three patients underwent surgery and further tumor immunohistochemistry. Imaging findings and immunohistochemical staining were compared with treatment outcomes. RESULTS: 18F-fallypride PET/MR was visually positive in 7 of 7 patients, and DRD2 target specificity could be confirmed by immunohistochemical staining. A significantly lower tracer standard uptake value (SUV) could be detected in the resistant patients (n = 3) than in the sensitive patients (n = 4; SUVmean, 4.67 ± 1.32 vs. 13.57 ± 2.42, p < 0.05). DRD2 expression determined by 18F-fallypride PET/MR corresponded with the DA treatment response. CONCLUSION: 18F-fallypride PET/MR may be a promising technique for predicting DA response in patients with prolactinoma.

Sellar Glomus Tumor Misdiagnosed as Pituitary Adenoma: A Case Report and Review of the Literature.

Glomus tumor is a rare mesenchymal tumor with an organ-like structure. Sellar glomus tumors are extremely rare with only six reported cases in the literature. Because of the lack of special clinical manifestations and imaging features, the disorder may be easily misdiagnosed as other sellar tumors, especially pituitary adenomas. Here, the present study showed a case of a 69-year-old male with hypopituitarism who was preliminarily misdiagnosed as non-functional pituitary adenoma.

TRIM65 determines the fate of a novel subtype of pituitary neuroendocrine tumors via ubiquitination and degradation of TPIT.

BACKGROUND: Pituitary neuroendocrine tumors (PitNETs) are common intracranial tumors that are classified into seven histological subtypes, including lactotroph, somatotroph, corticotroph, thyrotroph, gonadotroph, null cell, and plurihormonal PitNETs. However, the molecular characteristics of these types of PitNETs are not completely clear. METHODS: A total of 180 consecutive cases of PitNETs were collected to perform RNA sequencing. All subtypes of PitNETs were distinguished by unsupervised clustering analysis. We investigated the regulation of TPIT by TRIM65 and its effects on ACTH production and secretion in ACTH-secreting pituitary cell lines, as well as in murine models using biochemical analyses, confocal microscopy, and luciferase reporter assays. RESULTS: A novel subtype of PitNETs derived from TPIT lineage cells was identified as with normal TPIT transcription but with lowered protein expression. Furthermore, for the first time, TRIM65 was identified as the E3 ubiquitin ligase of TPIT. Depending on the RING domain, TRIM65 ubiquitinated and degraded the TPIT protein at multiple Lys sites. In addition, TRIM65-mediated ubiquitination of TPIT inhibited POMC transcription and ACTH production to determine the fate of the novel subtype of PitNETs in vitro and in vivo. CONCLUSION: Our studies provided a novel classification of PitNETs and revealed that the TRIM65-TPIT complex controlled the fate of the novel subtype of PitNETs, which provides a potential therapy target for Cushing's disease.

Case Report: Temozolomide Treatment of Refractory Prolactinoma Resistant to Dopamine Agonists.

Therapeutic agents for refractory prolactinomas that are resistant to dopamine agonists (DAs) are troublesome, and surgery often only removes a large part of the tumor without complete remission. Among the various second-line treatment regimens, the treatment effect of the alkylating agent temozolomide (TMZ) is only effective for approximately half of patients; however, complete remission is rare. Here we report a patient with prolactinoma who was resistant to high-dose cabergoline (CAB) treatment, demonstrating a continuous increase in both the tumor volume and the prolactin (PRL) level. Given that this case is a refractory prolactinoma, the patient underwent two transsphenoidal approach (TSA) surgeries. The pathological analysis indicated that the Ki-67 index increased significantly from 3% to 30%, and the expression levels of DRD2 and MGMT were low. Finally, TMZ treatment was recommended. A total of six cycles of TMZ standard chemotherapy shrank the tumor volume and the tumor disappeared completely. During the 6-month follow-up period, the tumor did not relapse again, and the PRL level was also normal. RNA sequencing and DNA whole genome sequencing were performed on this prolactinoma specimen, revealing 16 possible gene mutations, including a missense mutation of the PABPC1 gene. Additionally, the copy number variation analysis results showed that several chromosomes had copy number gains compared to the matched peripheral blood sample. In this case, low expression of DRD2 and high proliferation led to resistance to CAB, whereas low MGMT expression contributed to sensitivity to TMZ treatment. The results of genome sequencing still need further investigation at the molecular level to explain the tumor aggressiveness and high sensitivity to TMZ.

Proteomic Analysis of Perihematoma Tissue from Patients with Intracerebral Hemorrhage Using iTRAQ-Based Quantitative Proteomics.

Intracerebral hemorrhage is a complicated disorder with limited proven prognostic and therapeutic targets and elusive mechanisms. With proteomic methods, we aimed to explore the global protein expression profile of perihematomal tissue from ICH patients and identify potential pathophysiological pathways and protein markers. Using iTRAQ-labeling quantitative proteomics technology, four ICH brain sample and four non-ICH brain samples were analyzed. Among the 3740 quantifiable proteins, 884 were dysregulated in the patients compared to those in the controls (p < 0.05). After bioinformatics analysis, the differentially expressed proteins were found to be mostly involved in hemostatic processes, nutrient metabolism signaling pathways, and antioxidation pathways. Moreover, fibronectin 1 was revealed to be at the center of the protein-protein interaction networks. In summary, the potential pathways and brain protein markers that could potentially be used to predict the prognosis of ICH were obtained from the altered proteomic profile of perihematomal tissue. Thus, these data may yield novel insights into the mechanisms of ICH-induced secondary brain injury.

AEBP1 as a potential immune-related prognostic biomarker in glioblastoma: a bioinformatic analyses.

BACKGROUND: Adipocyte enhancer binding protein 1 (AEBP1) has been shown to be closely related to cancer progression; however research on its potential role in glioblastoma (GBM) remains limited. METHODS: Following an expression analysis of AEBP1 in GBM through the Oncomine database, other critical findings were accessed via The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) databases. Specifically, in addition to identifying differentially expressed genes, the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) were further investigated. Additionally, a gene set enrichment analysis (GSEA) was performed to examine the enrichment pathways in the AEBP1 high-expression group. To examine the prognostic role of AEBP1 in GBM, survival information was obtained from the Chinese Glioma Genome Atlas (CGGA) database. Finally, the relationship between the expression of AEBP1 and immune infiltration in GBM was examined by using the "Gene Module", "Survival Module", and "SCNA Module" on the website "Tumor Immune Estimation Resource (TIMER)". RESULTS: The Oncomine database revealed that AEBP1 was highly expressed in GBM. The prognostic analyses of 4 independent databases (i.e., TCGA, GTEx, Oncomine, and CGGA) revealed that AEBP1 was an independent predictable marker of GBM. The results of the GSEA showed that protein export, prion disease, cytokine receptor interaction, hematopoietic cell lineage, cell adhesion molecules, apoptosis, and the complement and coagulation cascades were differentially enriched in highly expressed AEBP1 phenotypes. Hence the conclusion is that the high expression of AEBP1 is closely correlated to poor prognosis of GBM. The immune analysis demonstrated that AEBP1 copy number alteration might affect immune infiltration in GBM tissues, and thus the survival outcomes of GBM patients. CONCLUSIONS: High AEBP1 expression in GBM is closely correlated to patient prognosis. AEBP1 is a potential therapeutic target for the inhibition of cancerous progression and the development of new immunotherapies for GBM.

The long noncoding RNA-H19/miRNA-93a/ATG7 axis regulates the sensitivity of pituitary adenomas to dopamine agonists.

Dopamine agonists (DAs), such as cabergoline and bromocriptine, are the first-line clinical treatment for prolactinomas. Our previous study demonstrated that long noncoding RNA H19 expression is frequently downregulated in human primary pituitary adenomas and is negatively correlated with tumor progression. However, the significance and mechanism of H19 in the DA treatment of prolactinomas are still unknown. In this study, we reported that H19 had a synergistic effect with DA treatment on prolactinomas in vitro and in vivo. Mechanistically, H19 promoted ATG7 expression in pituitary tumor cells by inhibiting miR-93a expression. In addition, a potential binding site between miR-93 and H19 was confirmed, and low expression of miR-93 was previously found in DA-resistant prolactinomas. Furthermore, we showed that miR-93a regulates ATG7 expression by targeting ATG7 mRNA. In conclusion, our study has identified the role of the H19-miR-93-ATG7 axis in DA treatment of prolactinomas, which may be a potential therapeutic target for human prolactinomas.

Ghrelin attenuates secondary brain injury following intracerebral hemorrhage by inhibiting NLRP3 inflammasome activation and promoting Nrf2/ARE signaling pathway in mice.

Ghrelin, a brain-gut peptide, has been proven to exert neuroprotection in different kinds of neurological diseases; however, its role and the potential molecular mechanisms in secondary brain injury (SBI) after intracerebral hemorrhage (ICH) are still unknown. In this study, we investigate whether treatment with ghrelin may attenuate SBI in a murine ICH model, and if so, whether the neuroprotective effects are due to the inhibition of nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation and promotion of nuclear factor-E2-related factor 2 (Nrf2)/antioxidative response element (ARE) signaling pathway. Stereotactically intrastriatal infusion of autologous blood was performed to mimic ICH. Ghrelin was given intraperitoneally immediately following ICH and again 1 h later. Results showed that ghrelin attenuated neurobehavioral deficits, brain edema, hematoma volume, and perihematomal cell death post-ICH. Ghrelin inhibited the NLRP3 inflammasome activation and subsequently suppressed the neuroinflammatory response as evidenced by reduced microglia activation, neutrophil infiltration, and pro-inflammatory mediators release after ICH. Additionally, ghrelin alleviated ICH-induced oxidative stress according to the chemiluminescence of luminol and lucigenin, malondialdehyde (MDA) content, and total superoxide dismutase (SOD) activity assays. These changes were accompanied by upregulation of Nrf2 expression, Nrf2 nuclear accumulation, and enhanced Nrf2 DNA binding activity, as well as by increased expressions of Nrf2 downstream target antioxidative genes, including NAD(P)H quinine oxidoreductase-1 (NQO1), glutathione cysteine ligase regulatory subunit (GCLC), and glutathione cysteine ligase modulatory subunit (GCLM). Together, our data suggested that ghrelin protected against ICH-induced SBI by inhibiting NLRP3 inflammasome activation and promoting Nrf2/ARE signaling pathway.

Evaluation of intestinal injury, inflammatory response and oxidative stress following intracerebral hemorrhage in mice.

Intestinal injury is a common complication following intracerebral hemorrhage (ICH), which leads to malnutrition, impaired immunity and unsatisfactory prognosis. Previous studies have revealed the pathogenesis of intestinal injury following traumatic brain injury using ischemic stroke models. However, the effects of ICH on intestinal injury remain unknown. The present study aimed to investigate the pathological alterations and molecular mechanism, as well as the time course of intestinal injury following ICH in mice. Male C57BL/6 mice were randomly divided into the following seven groups (n=6 mice/group): Control group, which underwent a sham operation, and six ICH groups (2, 6, 12 and 24 h, and days 3 and 7). The ICH model was induced by stereotactically injecting autologous blood in two stages into the brain. Subsequently, intestinal tissue was stained with hematoxylin and eosin for histopathological examination. Small intestinal motility was measured by charcoal meal test, and gut barrier dysfunction was evaluated by detecting the plasma levels of endotoxin. Quantitative polymerase chain reaction (qPCR), immunohistochemistry and ELISA analysis were performed to evaluate the mRNA and protein expression levels of inflammatory cytokines [interleukin (IL)­1ß, IL­6, tumor necrosis factor­α, intercellular adhesion molecule 1, monocyte chemotactic protein 1 and chemokine (C­C motif) ligand­5] in intestinal tissue and serum. Furthermore, intestinal leukocyte infiltration was detected by measuring myeloperoxidase activity. Oxidative stress was indirectly detected by measuring reactive oxygen species­associated markers (malondialdehyde content and superoxide dismutase activity assays) and the mRNA and protein expression levels of antioxidant genes [nuclear factor (erythroid­derived 2)­like 2, manganese superoxide dismutase and heme oxygenase 1] by qPCR and western blot analysis. The results demonstrated that significant destruction of the gut mucosa, delayed small intestinal motility, intestinal barrier dysfunction, and increased inflammatory responses and oxidative stress occurred rapidly in response to ICH. These symptoms occurred as early as 2 h after ICH and persisted for 7 days. These findings suggested that ICH may induce immediate and persistent damage to gut structure and barrier function, which may be associated with upregulation of inflammation and oxidative stress markers.

Downregulation of phosphoglycerate kinase 1 by shRNA sensitizes U251 xenografts to radiotherapy.

Phosphoglycerate kinase 1 (PGK1) has been demonstrated to be involved in radioresistance. The present study was designed to investigate the effect of PGK1 on the radioresistance in vivo. U251 glioma cells were transfected with the short hairpin RNA (shRNA)-PGK1 and pcDNA3.1-PGK1 using Lipofectamine 2000. The radiosensitivity of U251 xenografts was observed by tumor growth curve following radiotherapy. Quantitative PCR, western blot analysis and immunohistochemistry were performed to evaluate PGK1 expression in the xenografts from the different tumor models. The expression of PGK1 was maximally inhibited in response to shRNA4 at 24 h after the transfection in vitro. Tumor growth of the U251 xenografts was significantly inhibited following treatment with shRNA-PGK1 and radiotherapy. The expression of PGK1 in vivo at the mRNA and protein levels was downregulated by the treatment of shRNA1 when compared to levels following treatment with shNC and PBS after radiotherapy. The results showed that suppression of PGK1 enhanced the radiosensitivity of U251 xenografts and suggest that PGK1 may serve as a useful target in the treatment of radioresistant glioma.

Phosphoglycerate kinase 1 promotes radioresistance in U251 human glioma cells.

Phosphoglycerate kinase 1 (PGK1) has been found to be increased in radioresistant astrocytomas. The present study was designed to investigate the potential role of PGK1 in the radioresistance in U251 human cells. Quantitative PCR and western blot analysis were performed to evaluate PGK1 expression for mRNA levels and protein levels, respectively. The short hairpin RNA (shRNA)-PGK1 and the high expression plasmids were transfected to radioresistant U251 cells (RR-U251 cells) or normal U251 cells using lipofectamine™ 2000. The cell viability was determined by MTT assay. The wound-healing assay (WHA) was used to evaluate cell migration ability. Cell invasion abilities were examined using a Transwell culture chamber system. Our results showed that the expression of PGK1 was significantly increased in RR-U251 cells compared to normal U251 cells. Following irradiation, the cell viability as well as the migration and invasion ability were significantly higher in RR-U251 cells compared with normal U251 cells. Downregulating PGK1 using shRNA induced a significantly downregulated cell viability and decreased migration and invasion ability, and overexpression of PGK1 contributed to upregulated cell viability and increased migration and invasion ability, both in RR-U251 cells and normal U251 cells. These findings suggest that PGK1 could promote radioresistance in U251 human cells.

Mechanisms underlying the induction of the putative human tumor suppressor GPRC5A by retinoic acid.

Retinoic acid regulates the expression of genes involved in cell proliferation, differentiation and survival by direct control of gene transcription via activation of nuclear retinoid receptors bound to response elements in the promoters of target genes or by indirect mechanisms. Herein, we investigated the mechanism by which retinoic acid induces the expression of the human tumor suppressor GPRC5A. The proximal 5' upstream region of the GPRC5A gene was found to contain two potential RAR/RXR binding sites (RAREs) and one VDR/RXR binding site with direct repeat 5 (DR5) motifs designated DR5I (-489 to -473), DR5II (-136 to -120) and DR5III (-81 to -65). DR5II and DR5III but not DR5I were conserved among vertebrates. However, only DR5III (5'-TGT CCC TCT GCT CAC CC-3') was found to be the functional RARE for mediating induction of GPRC5A as indicated by electrophoretic mobility shift assay using wild type and mutated synthetic oligonucleotides representing different fragments of the promoter for competition with retinoic acid receptor beta RARE. Chromatin immunoprecipitation assay confirmed the binding of retinoic acid receptors alpha and gamma and retinoid X receptors alpha and beta to DR5III in intact cells. These results demonstrate the importance of functional analysis for validating the activity of predicted response elements.