Current updates relating to treatment for interstitial cystitis/bladder pain syndrome: systematic review and network meta-analysis.

BACKGROUND: Despite the publication of several meta-analyses regarding the efficacy of certain therapies in helping individuals with interstitial cystitis (IC) / bladder pain syndrome (BPS), these have not provided a comprehensive review of therapeutic strategies. The study aimed to determine the efficacy of various therapies for IC/BPS and identify potential moderating factors using randomized controlled trials (RCTs). METHODS: We queried the PubMed, Cochrane, and Embase databases to identify prospective RCTs using inclusion criteria: 1) patients diagnosed with IC, 2) interventions included relevant treatments, 3) comparisons were a specified control or placebo, 4) outcomes were mean differences for individual symptoms and structured questionnaires. The pairwise meta-analysis and network meta-analysis (NMA) were performed to compare the treatments used in IC/BPS. Hedges' g standardized mean differences (SMDs) were used for improvement in all outcomes using random-effects models. Efficacy outcomes included individual symptoms such as pain, frequency, urgency, and nocturia, as well as structured questionnaires measuring IC/BPS symptoms. RESULTS: A comprehensive literature search was conducted which identified 70 RCTs with 3,651 patients. The analysis revealed that certain treatments, such as instillation and intravesical injection, showed statistically significant improvements in pain and urgency compared to control or placebo groups in traditional pairwise meta-analysis. However, no specific treatment demonstrated significant improvement in all outcomes measured in the NMA. The results of moderator analyses to explore influential variables indicated that increasing age was associated with increased nocturia, while longer follow-up periods were associated with decreased frequency. CONCLUSION: This systematic review and meta-analysis provide insights into the efficacy of various treatments for IC. Current research suggests that a combination of therapies may have a positive clinical outcome for patients with IC, despite the fact that treatment for this condition is not straightforward. TRIAL REGISTRATION: PROSPERO CRD42022384024.

The Correlations between the Intensity of Histopathological Ubiquitin-Specific Protease 11 Staining and Progression of Prostate Cancer.

BACKGROUND: Ubiquitin-specific protease 11 (USP11), one of the principal phosphatase and tensin homolog (PTEN) deubiquitinases, can reserve PTEN polyubiquitination to maintain PTEN protein integrity and inhibit PI3K/AKT pathway activation. The aim of the current study was to investigate the associations between immunohistochemical USP11 staining intensities and prognostic indicators in individuals with prostate cancer. METHODS: Tissue microarrays (TMAs) were performed for human prostate cancer and normal tissue (control) samples. Data on patient's age, Gleason score, plasma prostate-specific antigen (PSA) titer, disease stage, and presence of seminal vesicles, lymph nodes, and surgical margin involvement were collected. A pathologist who was blinded to the clinical outcome data scored the TMA for USP11 staining intensity as either positive or negative. RESULTS: Cancerous tissues exhibited lower USP11 staining intensity, whereas the neighboring benign peri-tumoral tissues showed higher USP11 staining intensity. The degree of USP11 staining intensity was lower in patients with a higher PSA titer, higher Gleason score, or more advanced disease stage. Patients who showed positive USP11 staining were more likely to have more optimal clinical and biochemical recurrence-free survival statistics. CONCLUSIONS: USP11 staining intensity in patients with prostate cancer is negatively associated with several prognostic factors such as an elevated PSA titer and a high Gleason score. It also reflects both biochemical and clinical recurrence-free survival in such patients. Thus, USP11 staining is a valuable prognostic factor in patients with prostate cancer.

Efficacy of surgical treatment for post-prostatectomy urinary incontinence: a systematic review and network meta-analysis.

OBJECTIVES: Post-prostatectomy urinary incontinence (PPUI) is a major complication that reduces the quality of life in patients undergoing prostatectomy for benign prostatic hyperplasia and prostate cancer. However, there are currently limited guidelines on which surgical techniques are preferred after conservative treatment for PPUI. In this study, a systematic review and network meta-analysis (NMA) that can help determine the priority for the selection of surgical methods were performed. MATERIALS AND METHODS: We retrieved data from electronic literature searches of PubMed and the Cochrane Library through August 2021. We searched for randomized controlled trials studies on the surgical treatment of PPUI after surgery for benign prostatic hyperplasia or prostate cancer and included the terms artificial urethral sphincter (AUS), adjustable sling, nonadjustable sling, and injection of the bulking agent.The NMA pooled the odds ratios and 95% credible intervals (CrIs) using the number of patients achieving urinary continence, weight of pads used per day, number of pads used per day, and the International Consultation on Incontinence Questionnaire score. The therapeutic effect of each intervention on PPUI was compared and ranked using the surface under the cumulative ranking curve. RESULTS: A final 11 studies, including 1116 participants, were included in our NMA. The pooled overall odds ratios of patients achieving urinary continence compared with no treatment was 3.31 (95% CrI: 0.749, 15.710) in AUS, 2.97 (95% CrI: 0.412, 16.000) in adjustable sling, 2.33 (95% CrI: 0.559, 8.290) in nonadjustable sling, and 0.26 (95% CrI: 0.025, 2.500) in injection of bulking agent. In addition, this study shows the surface under the cumulative ranking curve values of ranking probabilities for each treatment performance, which indicated that AUS ranked first in terms of continence rate, International Consultation on Incontinence Questionnaire, pad weight, and pad use count. CONCLUSION: The results of this study suggested that only AUS had a statistically significant effect compared to the nontreatment group and the highest PPUI treatment effect ranking among other surgical treatments.

The correlation between the expression of ubiquitin-conjugating enzyme 2C and prostate cancer prognosis.

PURPOSE: Ubiquitin-conjugating enzyme E2 C (UBE2C) is known to show a causal relationship with cancer development and advancement. The role of UBE2C is to control the mitotic spindle checkpoint. Excess UBE2C has been identified in patients with advanced prostate cancer. The objective of the present study was to examine positive connections between the expression of UBE2C and prognostic factors for prostate cancer. METHODS: Prostate cancer patients' clinical data were analysed. Tissue microarrays (TMAs) were also performed for human prostate cancer tissues (n = 335) and adjacent non-neoplastic tissues (n = 22). TMA slides were incubated with antibodies against UBE2C. Cores were scored by a pathologist who was blind to cancer results. RESULTS: Of 335 prostate cancer patients, 200 could be assessed for biochemical recurrence, clinical recurrence, and overall survival. Human prostate cancer tissues showed higher expression of UBE2C than adjacent non-neoplastic tissues. High expression level of UBE2C showed a strong positive relationship with a high prostate-specific antigen (PSA), Gleason's score, and pathological stage of prostate cancer. Patients with a higher UBE2C grade demonstrated greater lymphatic engagement of prostate cancer than those with a lower UBE2C grade. CONCLUSION: The expression of UBE2C has positive correlations with several prognostic factors for prostate cancer. Thus, investigating the expression level of UBE2C staining is a promising tool for predicting prostate cancer prognosis.

Relationship Between Androgen Deprivation Therapy for Prostate Cancer and Risk of SARS-CoV-2 Infection: A Systematic Review and Meta-Analysis.

BACKGROUND: Several cohort studies have explored the relationship between androgen deprivation therapy (ADT) and the severity of coronavirus disease 2019 (COVID-19). This study aimed to characterize the relationship between ADT and the severity of COVID-19 in patients with prostate cancer. METHODS: A systematic search was conducted using PubMed, Embase, and Cochrane Library databases from the inception of each database until February 31, 2020. Patients with prostate cancer who were treated with ADT were assigned to treatment group while those patients who were not treated with ADT were assigned to the control group. Outcomes were severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) positivity, hospitalization, intensive care unit (ICU) admission, and death. The risk of bias was evaluated using ROBINS-I (Risk Of Bias In Non-randomized Studies of Interventions) tool. RESULTS: Three studies with qualitative synthesis were included. Finally, two studies with quantitative synthesis having a total of 44,213 patients were included for the present systematic review. There was no significant difference in SARS-CoV-2 positive rate (odds ratio [OR], 0.52; 95% confidence intervals [Cis], 0.13-2.09; P = 0.362), hospitalization (OR, 0.52; 95% CIs, 0.07-3.69; P = 0.514), ICU admission (OR, 0.93; 95% CIs, 0.39-2.23, P = 0.881), or death (OR, 0.88; 95% CIs, 0.06-12.06; P = 0.934) between ADT and non-ADT groups. CONCLUSION: Qualitative and quantitative analyses of previous studies revealed no significant effect of ADT on COVID-19. However, more studies with higher quality that explore biochemical and immunological factors involved are needed to confirm this finding in the future.

Microbiome in Bladder Cancer: A Systematic Review.

Although many studies on bladder cancer and the microbiome have been conducted so far, useful strains at the species level have not yet been identified. In addition, in the case of urine studies, methodological heterogeneity is too great, and in tissue studies, the species level through shotgun analysis has not been revealed, and studies using stool samples have provided only limited information. In this review, we will review all the microbiome studies related to bladder cancer so far through a systematic review.

Prostate cancer specific mortality after 5α-reductase inhibitors medication in benign prostatic hyperplasia patients: systematic review and meta-analysis.

BACKGROUND: Our study was conducted to investigate the effect of 5-ARI on the death rate from prostate cancer by means of an updated meta-analysis using related data. METHODS: We did comprehensive literature searches using the PubMed, Embase, and Cochrane databases through July 2019. We evaluated the risk of bias in such studies using the ROBINS-I tool and analyzed deaths from cancer and deaths from all causes using HR. RESULTS: The meta-analysis included 11 studies. The pooled overall HRs for cancer-specific deaths between 5-ARI treatment versus non-exposed groups were 0.937 (95% CI: 0.730, 1.201). In the subgroup moderator analysis, the number of patients and additional medication variables were significantly associated with deaths from all causes (p = 0.022 and p = 0.005, respectively). For detecting the publication bias or small-study effect in the included studies, we performed Begg and Mazumdar's correlation test (0.851 and 0.573) and Egger's regression coefficient test (0.035 and 0.245) and ​suggested that there was no evidence of publication bias or small-study effect in this meta-analysis. CONCLUSIONS: Our study indicated that exposure to 5-ARI had no close association between the overall death rate or cancer-specific deaths. To confirm these results, well-designed prospective studies with large samples are required.

The Positive Correlations between the Expression of Histopathological Ubiquitin-Conjugating Enzyme 2O Staining and Prostate Cancer Advancement.

BACKGROUND: The mTOR signaling pathway is inactivated by AMPK's tumor-suppressing function. It is recognized that ubiquitin conjugating enzyme 2O (UBE2O), which directly targets AMPK for ubiquitination and degradation, is intensified in human cancers. METHODS: This study investigated the clinical data about prostate cancer. Examination was also carried out into tissue microarrays (TMA) of human prostate cancer (n = 382) and adjacent non-neoplastic tissues around prostate cancer (n = 61). The TMA slides were incubated with antibodies against UBE2O, and the cores were scored by the pathologist blind to cancer results. RESULTS: Very strong positive correlations were identified between the expression of UBE2O staining and high PSA and pathological stage of prostate cancer. Cox's proportional hazard analysis established correlations between the following: (1) positive surgical margin and biochemical recurrence free survival, (2) PSA grade and clinical recurrence free survival, (3) regional lymph node positive and clinical recurrence free survival, (4) adjuvant treatment and overall survival, and (5) pathological T stage and overall survival. CONCLUSION: There is a positive correlation between the expression of UBE2O staining and prognosis for prostate cancer. Thus, a prostate cancer prognosis can be assessed with the expression of UBE2O staining.

Diagnostic accuracy of Raman spectroscopy for prostate cancer: a systematic review and meta-analysis.

BACKGROUND: Although various studies have been conducted to demonstrate the possibility of Raman spectroscopy (RS) as a diagnostic tool for prostate cancer (PC), it is difficult to use it in the real clinical area because of imitations in various research processes. Therefore, we did a systematic review and meta-analysis about the accuracy in diagnostic use of RS for PC. METHODS: A literature search was done using PubMed, Embase, and Cochrane library databases in March 2019 to analyze the accuracy of RS for diagnosis of PC. The accuracy of RS for diagnosis of PC was evaluated by means of pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and summary receiver operating characteristic (SROC). RESULTS: Five studies were included for qualitative analysis by screening the remaining articles according to the inclusion and exclusion criteria by means of a systematic review. The pooled sensitivity and specificity of RS were 0.89 (95% CI: 0.87-0.91) and 0.91 (95% CI: 0.89-0.93), respectively. The overall PLR and NLR were 9.12 (95% CI: 4.15-20.08) and 0.14 (95% CI: 0.07-0.29), respectively. The DOR of RS demonstrated high accuracy (73.32; 95% CI: 18.43-291.73). The area under the curves (AUCs) of SROC curves was 0.93. CONCLUSIONS: RS is an optical diagnostic method with high potential for diagnosis and grading of PC and has advantages of real-time and convenient use. In order to consider real-time use of RS in an actual clinical setting, more studies for standardization and generalization of RS performance and analytical method must be conducted.

Changes in diagnosis rate and treatment trends of benign prostatic hyperplasia in Korea: A nationwide population-based cohort study.

PURPOSE: This study aimed to evaluate benign prostatic hyperplasia (BPH) diagnosis rate and the changing landscape of medical and surgical management of BPH over the last decade using national population data of South Korea. METHODS: The present study analyzed data of patients diagnosed with BPH (N40) who underwent medical treatment or surgery in 3% of the national patient sample of the Health Insurance and Review Assessment database each year between 2012 and 2018. The primary outcome was the proportion of medical and surgical treatment for BPH. It was evaluated each year between 2012 and 2018. Secondary outcomes included total BPH diagnosis rate in each year of the study period. BPH diagnosis rate by age group was evaluated. The proportion of medical and surgical treatment for BPH according to the type of medical institution was investigated. RESULTS: The proportion of surgical treatment was 1.2% in 2012, 1.0% in 2013, 1.0% in 2014, 0.9% in 2015, 0.8% in 2016, 0.7% 2018, and 0.8% 2018, showing a progressive overall decrease from 2012 to 2018. The rate of surgical treatment for BPH increased with increasing age during the study period, showing a progressive overall increase (from 9,202 per 100,000 men in 2012 to 11,610 per 100 000 men in 2018). The number of patients with BPH was increased steadily from 2012 to 2018 in all age groups. The rate of surgical treatment in tertiary referral hospitals was the highest during the study period, followed by that in general hospitals, hospitals, and clinics. CONCLUSIONS: In Korea, the diagnosis rate of BPH was steadily increasing during the study period. Overall surgical treatment gradually decreased compared with an increase in medical treatment among all treatments for BPH. Thus, a comprehensive treatment plan for BPH should be established considering this trend.

Prevalence of benign pathology after partial nephrectomy for suspected renal tumor: A systematic review and meta-analysis.

OBJECTIVE: To investigate the overall prevalence of benign pathology after partial nephrectomy (PN) and identify predictive factors for benign pathology after PN. METHODS: A systematic review was performed following the PRISMA guidelines. PubMed/Medline, Embase, and the Cochrane Library were searched up to January 2019PRISMA guidelines. The data for the meta-analysis and network meta-analysis were pooled using a random-effects model. RESULTS: There were 144 studies included in the final analysis, which was comprised of 79 observational studies (n = 37,300) and 65 comparative studies (n = 18,552). The overall prevalence rate of benign pathology after PN was 0.19 (95% CI: 0.18-0.21). According to the procedure types, the prevalence rate of benign pathology was 0.17 (95% CI: 0.15-0.19), 0.24 (95% CI: 0.22-0.27), and 0.16 (95% CI: 0.15-0.18) in open partial nephrectomy, laparoscopic partial nephrectomy, and robot-assisted laparoscopic partial nephrectomy, respectively. The significant moderating factors were gender, publication year, the origin of the study, and procedure types. The three most common benign pathology types were oncocytomas, angiomyolipomas, and renal cysts (44.50%, 30.20%, and 10.99%, respectively). CONCLUSIONS: The overall prevalence of benign pathology after PN was not low and it was affected by female gender, studies published before 2010, studies originating from Western areas, and laparoscopic procedure types.

Bilateral ureteral replacement using ileum in bilateral refractory ureteral stricture with renal insufficiency: a case report and review of literature.

Although several studies have reported the efficacy and safety of ileal ureter in refractory ureteral strictures, reports on its long-term outcomes are still insufficient. In this case report, we present an excellent long-term outcome over three years without complication of bilateral ureteral replacement with ileum using '7' shaped configuration in a woman after undergoing radical hysterectomy and radiation therapy who had renal insufficiency due to ureteral stricture. This study gave us a useful information about the feasibility and safety of bilateral ileal ureter in patients with ureteral strictures and renal insufficiency.

Association between prostate-specific antigen and serum testosterone: A systematic review and meta-analysis.

BACKGROUND: Serum testosterone assays are an important tool in the clinical evaluation of a number of endocrine disorders including male hypogonadism. However, serum testosterone has a limited role in real clinical use due to its inaccuracy. We aimed to assess the association between prostate-specific antigen (PSA) and testosterone as well as the effects of various types of testosterone replacement therapy (TRT) for PSA level. METHODS: Two electronic databases were screened: PubMed (1966 through December 2018) and Cochrane Library (1993 through December 2018). The first strategy compared the overall increase in PSA following testosterone treatment compared with placebo. The second strategy analyzed the overall association between PSA and testosterone among the observational studies. RESULTS: In the first strategy, 22 articles were included in the final analysis. In the second strategy, 18 studies were included. Testosterone replacement therapy (TRT) showed a significant change in PSA level compared to that in the placebo group (mean difference [MD]: 0.13, 95% CI: 0.01-0.25, P = .04). Compared to placebo, only intramuscular (IM) TRT shows a significant change in PSA level group (MD: 0.16, 95% CI: 0.01-0.30, P = .04), as neither the oral nor topical type showed a significant change in PSA. In the second strategy analysis, there was no overall correlation found between PSA and testosterone (z = 0.04, 95% CI: -0.04 to 0.12, P = .04; r = 0.039). However, in the subgroup of non-BPH (benign prostate hyperplasia), a significant correlation between PSA and testosterone (z = 0.07, 95% CI: 0.01-0.13, P = .009; r = 0.089) was found. CONCLUSIONS: We found that TRT, particularly IM TRT, significantly changed the PSA level compared with the placebo group. Furthermore, there was a significant correlation between PSA and testosterone in patients with non-BPH. According to these findings, we suggest the possibility of PSA as a surrogate marker of testosterone.

Learning curve analysis of colorectal endoscopic submucosal dissection (ESD) for laterally spreading tumors by endoscopists experienced in gastric ESD.

BACKGROUND: Endoscopic submucosal dissection (ESD) for various colorectal neoplasms is more technically difficult than gastric ESD. We evaluated treatment outcomes and the learning curve for colorectal ESD of laterally spreading tumors (LSTs) based on the experience of a single endoscopist. METHODS: We included 93 colorectal ESD procedures for colorectal LST that were performed between March 2009 and June 2012 by a single experienced endoscopist who previously performed hundreds of cases of gastric ESD. The cases were grouped chronologically into three periods by multi-dimensional analyses. For procedure time, the learning curve was analyzed using the moving average method, and for complication, the learning curve was analyzed using cumulative sum (cusum) method. RESULTS: The median procedure time for 93 colorectal ESD was 45 min, and the rates of en bloc resection and R0 resection were 89.25 and 83.87 %. When results were compared among three periods in order to determine the learning curve, the procedure time and en bloc resection rates were not significantly different. However, the procedure proficiency (about 0.16 cm(2)/min) was significantly faster during the second period, after about 25 cases of colorectal ESD. In the third period (about 50 cases), the number and rate of en bloc resection (over 90 %) reached the same as that of en bloc R0 resection. When comparing outcomes based on LST subtype, the procedure proficiency of LST-granular type (LST-G) was significantly faster than that of LST-non granular type (LST-NG) (LST-NG, 0.072 cm(2)/min; LST-G, 0.157 cm(2)/min; p = 0.01). CONCLUSION: Endoscopists fully experienced in gastric ESD need a relatively short learning period for colorectal ESD in terms of procedure time and complication. However, approximately 50 cases might be needed to acquire an adequate skill of colorectal ESD for LST in an experienced gastric ESD endoscopist. Colorectal ESD for LST-NG seems to have higher technical difficulty and a longer learning curve than LST-G.

Prognostic factors for urachal cancer: a bayesian model-averaging approach.

PURPOSE: This study was conducted to evaluate prognostic factors and cancer-specific survival (CSS) in a cohort of 41 patients with urachal carcinoma by use of a Bayesian model-averaging approach. MATERIALS AND METHODS: Our cohort included 41 patients with urachal carcinoma who underwent extended partial cystectomy, total cystectomy, transurethral resection, chemotherapy, or radiotherapy at a single institute. All patients were classified by both the Sheldon and the Mayo staging systems according to histopathologic reports and preoperative radiologic findings. Kaplan-Meier survival curves and Cox proportional-hazards regression models were carried out to investigate prognostic factors, and a Bayesian model-averaging approach was performed to confirm the significance of each variable by using posterior probabilities. RESULTS: The mean age of the patients was 49.88 ± 13.80 years and the male-to-female ratio was 24:17. The median follow-up was 5.42 years (interquartile range, 2.8-8.4 years). Five- and 10-year CSS rates were 55.9% and 43.4%, respectively. Lower Sheldon (p=0.004) and Mayo (p<0.001) stage, mucinous adenocarcinoma (p=0.005), and larger tumor size (p=0.023) were significant predictors of high survival probability on the basis of a log-rank test. By use of the Bayesian model-averaging approach, higher Mayo stage and larger tumor size were significant predictors of cancer-specific mortality in urachal carcinoma. CONCLUSIONS: The Mayo staging system might be more effective than the Sheldon staging system. In addition, the multivariate analyses suggested that tumor size may be a prognostic factor for urachal carcinoma.

Thin sectioning of slice preparations for immunohistochemistry.

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue. Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle. Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm. We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections. The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed. We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice. This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice. A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT). This is then quickly frozen with crushed dry ice and the resulting wafer can be position easily for cryostat sectioning. Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

Stromal cell-derived inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells.

To induce differentiation of embryonic stem cells (ESCs) into specialized cell types for therapeutic purposes, it may be desirable to combine genetic manipulation and appropriate differentiation signals. We studied the induction of dopaminergic (DA) neurons from mouse ESCs by overexpressing the transcription factor Nurr1 and coculturing with PA6 stromal cells. Nurr1-expressing ESCs (N2 and N5) differentiated into a higher number of neurons (approximately twofold) than the naïve ESCs (D3). In addition, N2/N5-derived cells contained a significantly higher proportion (>50%) of tyrosine hydroxylase (TH)+ neurons than D3 (<30%) and an even greater proportion of TH+ neurons (approximately 90%) when treated with the signaling molecules sonic hedgehog, fibroblast growth factor 8, and ascorbic acid. N2/N5-derived cells express much higher levels of DA markers (e.g., TH, dopamine transporter, aromatic amino acid decarboxylase, and G protein-regulated inwardly rectifying K+ channel 2) and produce and release a higher level of dopamine, compared with D3-derived cells. Furthermore, the majority of generated neurons exhibited electrophysiological properties characteristic of midbrain DA neurons. Finally, transplantation experiments showed efficient in vivo integration/generation of TH+ neurons after implantation into mouse striatum. Taken together, our results show that the combination of genetic manipulation(s) and in vitro cell differentiation conditions offers a reliable and effective induction of DA neurons from ESCs and may pave the way for future cell transplantation therapy in Parkinson's disease.

A proximal promoter domain containing a homeodomain-binding core motif interacts with multiple transcription factors, including HoxA5 and Phox2 proteins, and critically regulates cell type-specific transcription of the human norepinephrine transporter gene.

Expression of the norepinephrine transporter (NET), which mediates the reuptake of norepinephrine into presynaptic nerve terminals, is restricted to noradrenergic (NA) neurons. We have demonstrated previously that the 9.0 kb upstream sequences and the first intron residing in the 5' untranslated area are critical for high-level and NA cell-specific transcription. Here, using transient transfection assays, we show that 4.0 kb of the 5' upstream sequences contains sufficient genetic information to drive reporter gene expression in an NA cell type-specific manner. Three functional domains appear to be potentially important for the regulation of human NET (hNET) gene transcription: an upstream enhancer region at -4.0 to -3.1 kb, a proximal domain at -133 to -75 bp, and a middle silencer region between these two domains. DNase I footprinting analysis of the proximal promoter region shows that a subdomain at -128 to -80 bp is protected in a cell-specific manner. We provide evidence that multiple protein factors interact with the proximal promoter domain to critically regulate the transcriptional activity of the hNET gene. In the middle of this proximal subdomain resides a homeodomain (HD)-binding core motif, which interacts with HD factors, including Phox2a and HoxA5, in an NA-specific manner. Cotransfection analyses suggest that HoxA5 and Phox2a may transactivate the hNET gene promoter. Together with previous studies indicating direct activation of dopamine beta-hydroxylase transcription by Phox2a/2b, the present results support a model whereby Phox2 proteins may coordinately regulate the phenotypic specification of NA neurons by activating both NA biosynthetic and reuptake genes.