Activation of Bivalent Gene POU4F1 Promotes and Maintains Basal-like Breast Cancer.

Basal-like breast cancer (BLBC) is the most aggressive molecular subtype of breast cancer with worse prognosis and fewer treatment options. The underlying mechanisms upon BLBC transcriptional dysregulation and its upstream transcription factors (TFs) remain unclear. Here, among the hyperactive candidate TFs of BLBC identified by bioinformatic analysis, POU4F1 is uniquely upregulated in BLBC and is associated with poor prognosis. POU4F1 is necessary for the tumor growth and malignant phenotypes of BLBC through regulating G1/S transition by direct binding at the promoter of CDK2 and CCND1. More importantly, POU4F1 maintains BLBC identity by repressing ERα expression through CDK2-mediated EZH2 phosphorylation and subsequent H3K27me3 modification in ESR1 promoter. Knocking out POU4F1 in BLBC cells reactivates functional ERα expression, rendering BLBC sensitive to tamoxifen treatment. In-depth epigenetic analysis reveals that the subtype-specific re-configuration and activation of the bivalent chromatin in the POU4F1 promoter contributes to its unique expression in BLBC, which is maintained by DNA demethylase TET1. Together, these results reveal a subtype-specific epigenetically activated TF with critical role in promoting and maintaining BLBC, suggesting that POU4F1 is a potential therapeutic target for BLBC.

HDAC2 Is Involved in the Regulation of BRN3A in Melanocytes and Melanoma.

The neural crest transcription factor BRN3A is essential for the proliferation and survival of melanoma cells. It is frequently expressed in melanoma but not in normal melanocytes or benign nevi. The mechanisms underlying the aberrant expression of BRN3A are unknown. Here, we investigated the epigenetic regulation of BRN3A in melanocytes and melanoma cell lines treated with DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC) inhibitors. DNMT and HAT inhibition did not significantly alter BRN3A expression levels, whereas panHDAC inhibition by trichostatin A led to increased expression. Treatment with the isoform-specific HDAC inhibitor mocetinostat, but not with PCI-34051, also increased BRN3A expression levels, suggesting that class I HDACs HDAC1, HDAC2, and HDAC3, and class IV HDAC11, were involved in the regulation of BRN3A expression. Transient silencing of HDACs 1, 2, 3, and 11 by siRNAs revealed that, specifically, HDAC2 inhibition was able to increase BRN3A expression. ChIP-Seq analysis uncovered that HDAC2 inhibition specifically increased H3K27ac levels at a distal enhancer region of the BRN3A gene. Altogether, our data suggest that HDAC2 is a key epigenetic regulator of BRN3A in melanocytes and melanoma cells. These results highlight the importance of epigenetic mechanisms in regulating melanoma oncogenes.

Genetic interplay between transcription factor Pou4f1/Brn3a and neurotrophin receptor Ret in retinal ganglion cell type specification.

BACKGROUND: While the transcriptional code governing retinal ganglion cell (RGC) type specification begins to be understood, its interplay with neurotrophic signaling is largely unexplored. In mice, the transcription factor Brn3a/Pou4f1 is expressed in most RGCs, and is required for the specification of RGCs with small dendritic arbors. The Glial Derived Neurotrophic Factor (GDNF) receptor Ret is expressed in a subset of RGCs, including some expressing Brn3a, but its role in RGC development is not defined. METHODS: Here we use combinatorial genetic experiments using conditional knock-in reporter alleles at the Brn3a and Ret loci, in combination with retina- or Ret specific Cre drivers, to generate complete or mosaic genetic ablations of either Brn3a or Ret in RGCs. We then use sparse labelling to investigate Brn3a and Ret gene dosage effects on RGC dendritic arbor morphology. In addition, we use immunostaining and/or gene expression profiling by RNASeq to identify transcriptional targets relevant for the potential Brn3a-Ret interaction in RGC development. RESULTS: We find that mosaic gene dosage manipulation of the transcription factor Brn3a/Pou4f1 in neurotrophic receptor Ret heterozygote RGCs results in altered cell fate decisions and/or morphological dendritic defects. Specific RGC types are lost if Brn3a is ablated during embryogenesis and only mildly affected by postnatal Brn3a ablation. Sparse but not complete Brn3a heterozygosity combined with complete Ret heterozygosity has striking effects on RGC type distribution. Brn3a only mildly modulates Ret transcription, while Ret knockouts exhibit slightly skewed Brn3a and Brn3b expression during development that is corrected by adult age. Brn3a loss of function modestly but significantly affects distribution of Ret co-receptors GFRα1-3, and neurotrophin receptors TrkA and TrkC in RGCs. CONCLUSIONS: Based on these observations, we propose that Brn3a and Ret converge onto developmental pathways that control RGC type specification, potentially through a competitive mechanism requiring signaling from the surrounding tissue.

The Retinal Ganglion Cell Response to Blast-Mediated Traumatic Brain Injury Is Genetic Background Dependent.

Purpose: The purpose of this study was to examine the influence of genetic background on the retinal ganglion cell (RGC) response to blast-mediated traumatic brain injury (TBI) in Jackson Diversity Outbred (J:DO), C57BL/6J and BALB/cByJ mice. Methods: Mice were subject to one blast injury of 137 kPa. RGC structure was analyzed by optical coherence tomography (OCT), function by the pattern electroretinogram (PERG), and histologically using BRN3A antibody staining. Results: Comparison of the change in each group from baseline for OCT and PERG was performed. There was a significant difference in the J:DOΔOCT compared to C57BL/6J mice (P = 0.004), but not compared to BALB/cByJ (P = 0.21). There was a significant difference in the variance of the ΔOCT in J:DO compared to both C57BL/6J and BALB/cByJ mice. The baseline PERG amplitude was 20.33 ± 9.32 µV, which decreased an average of -4.14 ± 12.46 µV following TBI. Baseline RGC complex + RNFL thickness was 70.92 ± 4.52 µm, which decreased an average of -1.43 ± 2.88 µm following blast exposure. There was not a significant difference in the ΔPERG between J:DO and C57BL/6J (P = 0.13), although the variances of the groups were significantly different. Blast exposure in J:DO mice results in a density change of 558.6 ± 440.5 BRN3A-positive RGCs/mm2 (mean ± SD). Conclusions: The changes in retinal outcomes had greater variance in outbred mice than what has been reported, and largely replicated herein, for inbred mice. These results demonstrate that the RGC response to blast injury is highly dependent upon genetic background.

Biomarkers of lesion severity in a rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION).

The rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION) is similar in many of its pathophysiological responses to clinical NAION. Like human NAION, there is significant variability in the severity of the lesion produced, and little is known of the parameters associated with rNAION induction severity or if pre- or early post-induction biomarkers can be identified that enable prediction of lesion severity and ultimate loss of retinal ganglion cells (RGCs). Adult male Sprague-Dawley outbred rats were evaluated for various parameters including physiological characteristics (heart rate, respiratory rate, temperature, hematocrit [Hct]), optic nerve head (ONH) appearance, pre- and post-induction mean diameter, and intravenous fluorescein and indocyanine green angiographic patterns of vascular leakage at 5 hours post-induction, performed using a spectral domain-optical coherence tomography (SD-OCT) instrument. Early changes were correlated with ultimate RGC loss by Brn3a (+) immunohistology. RGC loss also was correlated with the relative level of laser exposure. The severity of ONH edema 2d, but not 5hr, post induction was most closely associated with the degree of RGC loss, revealing a threshold effect, and consistent with a compartment syndrome where a minimum level of capillary compression within a tight space is responsible for damage. RGC loss increased dramatically as the degree of laser exposure increased. Neither physiological parameters nor the degree of capillary leakage 5hr post induction were informative as to the ultimate degree of RGC loss. Similar to human NAION, the rNAION model exhibits marked variability in lesion severity. Unlike clinical NAION, pre-induction ONH diameter likely does not contribute to ultimate lesion severity; however, cross-sectional ONH edema can be used as a biomarker 2d post-induction to determine randomization of subjects prior to inclusion in specific neuroprotection or neuroregeneration studies.

Haploinsufficiency of POU4F1 causes an ataxia syndrome with hypotonia and intention tremor.

De novo, heterozygous, loss-of-function variants were identified in Pou domain, class 4, transcription factor 1 (POU4F1) via whole-exome sequencing in four independent probands presenting with ataxia, intention tremor, and hypotonia. POU4F1 is expressed in the developing nervous system, and mice homozygous for null alleles of Pou4f1 exhibit uncoordinated movements with newborns being unable to successfully right themselves to feed. Head magnetic resonance imaging of the four probands was reviewed and multiple abnormalities were noted, including significant cerebellar vermian atrophy and hypertrophic olivary degeneration in one proband. Transcriptional activation of the POU4F1 p.Gln306Arg protein was noted to be decreased when compared with wild type. These findings suggest that heterozygous, loss-of-function variants in POU4F1 are causative of a novel ataxia syndrome.

Overexpression of D-amino acid oxidase prevents retinal neurovascular pathologies in diabetic rats.

AIMS/HYPOTHESIS: Diabetic retinopathy is characterised by retinal neurodegeneration and retinal vascular abnormalities, affecting one third of diabetic patients with disease duration of more than 10 years. Accumulated evidence suggests that serine racemase (SR) and D-serine are correlated with the pathogenesis of diabetic retinopathy and the deletion of the Srr gene reverses neurovascular pathologies in diabetic mice. Since D-serine content is balanced by SR synthesis and D-amino acid oxidase (DAAO) degradation, we examined the roles of DAAO in diabetic retinopathy and further explored relevant therapy. METHODS: Rats were used as a model of diabetes by i.p. injection of streptozotocin at the age of 2 months and blood glucose was monitored with a glucometer. Quantitative real-time PCR was used to examine Dao mRNA and western blotting to examine targeted proteins in the retinas. Bisulphite sequencing was used to examine the methylation of Dao mRNA promoter in the retinas. Intravitreal injection of DAAO-expressing adenovirus (AAV8-DAAO) was conducted one week before streptozotocin administration. Brain specific homeobox/POU domain protein 3a (Brn3a) immunofluorescence was conducted to indicate retinal ganglion cells at 3 months after virus injection. The permeability of the blood-retinal barrier was examined by Evans blue leakage from retinal capillaries. Periodic acid-Schiff staining and haematoxylin counterstaining were used to indicate retinal vasculature, which was further examined with double immunostaining at 7 months after virus injection. RESULTS: At the age of 12 months, DAAO mRNA and protein levels in retinas from diabetic animals were reduced to 66.2% and 70.4% of those from normal (control) animals, respectively. The Dao proximal promoter contained higher levels of methylation in diabetic than in normal retinas. Consistent with the observation, DNA methyltransferase 1 was increased in diabetic retinas. Injection of DAAO-expressing virus completely prevented the loss of retinal ganglion cells and the disruption of blood-retinal barrier in diabetic rats. Diabetic retinas contained retinal ganglion cells at a density of 54 ± 4/mm2, which was restored to 68 ± 9/mm2 by DAAO overexpression, similar to the levels in normal retinas. The ratio between the number of endothelial cells and pericytes in diabetic retinas was 6.06 ± 1.93/mm2, which was reduced to 3.42 ± 0.55/mm2 by DAAO overexpression; the number of acellular capillaries in diabetic retinas was 10 ± 5/mm2, which was restored to 6 ± 2/mm2 by DAAO overexpression, similar to the levels in normal retinas. Injection of the DAAO-expressing virus increased the expression of occludin and reduced gliosis, which were examined to probe the mechanism by which the disrupted blood-retinal barrier in diabetic rats was rescued and retinal neurodegeneration was prevented. CONCLUSIONS/INTERPRETATION: Altogether, overexpression of DAAO before the onset of diabetes protects against neurovascular abnormalities in retinas from diabetic rats, which suggests a novel strategy for preventing diabetic retinopathy. Graphical abstract.

Remote Ischemic Post-Conditioning Therapy is Protective in Mouse Model of Traumatic Optic Neuropathy.

Traumatic optic neuropathy (TON) is characterized by visual dysfunction after indirect or direct injury to the optic nerve following blunt head trauma. TON is associated with increased oxidative stress and inflammation resulting in retinal ganglion cell (RGC) death. Remote ischemic post-conditioning (RIC) has been shown to enhance endogenous protective mechanisms in diverse disease models including stroke, vascular cognitive impairment (VCI), retinal injury and optic nerve injury. However, the protective mechanisms underlying the improvement of retinal function and RGC survival after RIC treatment remain unclear. Here, we hypothesized that RIC therapy may be protective following TON by preventing RGC death, oxidative insult and inflammation in the mouse retina. To carry out the study, mice were divided in three different groups (Control, TON and TON + RIC). We harvested retinal tissue 5 days after TON induction for western blotting and histochemical analysis. We observed increased TON-induced retinal cell death compared with controls by cleaved caspase-3 immunohistochemistry. Furthermore, the TON cohort demonstrated increased TUNEL positive cells which were significantly attenuated by RIC. Immunofluorescence data showed that oxidative stress markers dihydroethidium (DHE), NOX-2 and nitrotyrosine expression were elevated in the TON group relative to controls and RIC therapy significantly reduced the expression level of these markers. Next, we found that the proinflammatory cytokine TNF-α was increased and anti-inflammatory IL-10 was decreased in plasma of TON animals, and RIC therapy reversed this expression level. Interestingly, western blotting of retinal tissue showed that RGC marker Brn3a and tight junction proteins (ZO-1 and Occludin), and AMPKα1 expression were downregulated in the TON group compared to controls. However, RIC significantly increased the expression levels of these proteins. Together these data suggest that RIC therapy activates endogenous protective mechanisms which may attenuate TON-induced oxidative stress and inflammation, and improves BRB integrity.

Analysis of DNA methylation-driven genes for predicting the prognosis of patients with colorectal cancer.

Aberrant promoter methylation and ensuing abnormal gene expression are important epigenetic mechanisms that contribute to colorectal oncogenesis. Yet, the prognostic significance of such methylation-driven genes in colorectal cancer (CRC) remains obscure. Herein, a total of 181 genes were identified as the methylation-driven molecular features of CRC by integrated analysis of the expression profiles and the matched DNA methylation data from The Cancer Genome Atlas (TCGA) database. Among them, a five-gene signature (POU4F1, NOVA1, MAGEA1, SLCO4C1, and IZUMO2) was developed as a risk assessment model for predicting the clinical outcomes in CRC. The Kaplan-Meier analysis and Harrell's C index demonstrated that the risk assessment model significantly distinguished the patients in high or low-risk groups (p-value < 0.0001 log-rank test, HR: 2.034, 95% CI: 1.419-2.916, C index: 0.655). The sensitivity and specificity were validated by the receiver operating characteristic (ROC) analysis. Furthermore, different pharmaceutical treatment responses were observed between the high-risk and low-risk groups. Indeed, the methylation-driven gene signature could act as an independent prognostic evaluation biomarker for assessing the OS of CRC patients and guiding the pharmaceutical treatment. Compared with known biomarkers, the methylation-driven gene signature could reveal cross-omics molecular features for improving clinical stratification and prognosis.

GFI1 functions to repress neuronal gene expression in the developing inner ear hair cells.

Despite the known importance of the transcription factors ATOH1, POU4F3 and GFI1 in hair cell development and regeneration, their downstream transcriptional cascades in the inner ear remain largely unknown. Here, we have used Gfi1cre;RiboTag mice to evaluate changes to the hair cell translatome in the absence of GFI1. We identify a systematic downregulation of hair cell differentiation genes, concomitant with robust upregulation of neuronal genes in the GFI1-deficient hair cells. This includes increased expression of neuronal-associated transcription factors (e.g. Pou4f1) as well as transcription factors that serve dual roles in hair cell and neuronal development (e.g. Neurod1, Atoh1 and Insm1). We further show that the upregulated genes are consistent with the NEUROD1 regulon and are normally expressed in hair cells prior to GFI1 onset. Additionally, minimal overlap of differentially expressed genes in auditory and vestibular hair cells suggests that GFI1 serves different roles in these systems. From these data, we propose a dual mechanism for GFI1 in promoting hair cell development, consisting of repression of neuronal-associated genes as well as activation of hair cell-specific genes required for normal functional maturation.

POU4F1 confers trastuzumab resistance in HER2-positive breast cancer through regulating ERK1/2 signaling pathway.

Over-expression of the human epidermal growth factor receptor-2 (HER2) is related to aggressive tumors and poor prognosis in breast cancer. Trastuzumab (TRA) resistance leads to tumor recurrence and metastasis, resulting in poor prognosis in HER2-positive breast cancer. POU Class 4 Homeobox 1 (POU4F1) is a member of the POU domain family transcription factors, and has a key role in regulating cancers. However, its effects on TRA-resistant HER2-positive breast cancer are still vague. In the present study, we found that POU4F1 expression was dramatically increased in clinical breast cancer specimens with TRA resistance. Higher POU4F1 was also detected in HER2-positive breast cancer cells with TRA resistance than that of the parental ones. Poor prognosis was detected in breast cancer patients with high POU4F1 expression. Under TRA treatment, POU4F1 knockdown significantly reduced the proliferative capacity of HER2-positive breast cancer cells with TRA resistance. POU4F1 silence also sensitized resistant HER-positive breast cancer cells to TRA treatment in vivo using a xenograft mouse model, along with the markedly reduced tumor growth rate and tumor weight. Moreover, we found that POU4F1 deletion greatly decreased the activation of mitogen-activated or extracellular signal-regulated protein kinase kinases 1 and 2 (MEK1/2) and extracellular-regulated kinase 1/2 (ERK1/2) signaling pathways in breast cancer cells with TRA resistance. Migration and invasion were also effectively hindered by POU4F1 knockdown in TRA-resistant HER2-positive breast cancer cells. Notably, we found that POU4F1 deletion-improved chemosensitivity of HER2-positive breast cancer cells with drug-resistance to TRA treatment was closely associated with the blockage of ERK1/2 signaling. Collectively, our findings reported a critical role of POU4F1 in regulating TRA resistance, and demonstrated the underlying molecular mechanisms in HER2-positive breast cancer. Thus, POU4F1 may be a promising prognostic and therapeutic target to develop effective treatment for overcoming TRA resistance.

Neural transcription factor Pou4f1 promotes renal fibrosis via macrophage-myofibroblast transition.

Unresolved inflammation can lead to tissue fibrosis and impaired organ function. Macrophage-myofibroblast transition (MMT) is one newly identified mechanism by which ongoing chronic inflammation causes progressive fibrosis in different forms of kidney disease. However, the mechanisms underlying MMT are still largely unknown. Here, we discovered a brain-specific homeobox/POU domain protein Pou4f1 (Brn3a) as a specific regulator of MMT. Interestingly, we found that Pou4f1 is highly expressed by macrophages undergoing MMT in sites of fibrosis in human and experimental kidney disease, identified by coexpression of the myofibroblast marker, α-SMA. Unexpectedly, Pou4f1 expression peaked in the early stage in renal fibrogenesis in vivo and during MMT of bone marrow-derived macrophages (BMDMs) in vitro. Mechanistically, chromatin immunoprecipitation (ChIP) assay identified that Pou4f1 is a Smad3 target and the key downstream regulator of MMT, while microarray analysis defined a Pou4f1-dependent fibrogenic gene network for promoting TGF-ß1/Smad3-driven MMT in BMDMs at the transcriptional level. More importantly, using two mouse models of progressive renal interstitial fibrosis featuring the MMT process, we demonstrated that adoptive transfer of TGF-ß1-stimulated BMDMs restored both MMT and renal fibrosis in macrophage-depleted mice, which was prevented by silencing Pou4f1 in transferred BMDMs. These findings establish a role for Pou4f1 in MMT and renal fibrosis and suggest that Pou4f1 may be a therapeutic target for chronic kidney disease with progressive renal fibrosis.

Toll-like receptor-9 (TLR-9) deficiency alleviates optic nerve injury (ONI) by inhibiting inflammatory response in vivo and in vitro.

Traumatic optic neuropathy is a common clinical problem. Damage to the optic nerve leads to shear stress and triggers secondary swelling within the optic canal. The study aims to explore the role of the inflammatory response following optic nerve injury (ONI) in toll-like receptor-9 knockout mice (TLR-9-/-) compared to wild-type mice (WT). At first, TLR-9-/- and WT mice were subjected to ONI. We then found that ONI significantly up-regulated TLR-9 expression levels in retinal tissues of WT mice. The retinal degeneration after ONI was alleviated in TLR-9-/- mice, as evidenced by the increased number of retinal ganglion cells (RGCs) and thickness of inner retinal layer (IRL). TUNEL staining and immunofluorescence staining of BRN3A indicated that TLR-9 knockout effectively improved the survival of RGCs. ONI-enhanced expression of Iba-1 and TMEM119 was markedly reduced in TLR-9-/- mice, indicating the suppression of microglial activation. Moreover, production of pro-inflammatory regulators, including inducible nitric oxide synthase (iNOS), macrophage chemo-attractant protein (MCP)-1, cyclooxygenase-2 (COX-2), interleukin (IL)-1ß, IL-18 and tumor necrosis factor-α (TNF-α), was significantly decreased in TLR-9-/- mice following ONI. TLR-9 knockout-attenuated inflammation was mainly through repressing myeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase 4 (IRAK4). Furthermore, ONI greatly up-regulated the protein expression levels of phosphorylated (p)-IKKα, p-IκBα and p-nuclear factor (NF)-κB, whereas being repressed in TLR-9-/- mice. The effects of TLR-9 on ONI were verified in lipopolysaccharide (LPS)-stimulated retinal microglial cells transfected with small interfering RNA TLR-9 (siTLR-9). As expected, promoting TLR-9 with its agonist markedly restored inflammation in TLR-9 knockdown cells stimulated by LPS. Therefore, all findings above suggested that suppressing TLR-9 showed neuroprotective effects against ONI through reducing inflammatory response, and TILR-9 might be a promising therapeutic target to develop effective strategies for the treatment of optic neuropathies.

No association between POU4F1, POU4F2, ISL1 polymorphisms and normal-tension glaucoma.

BACKGROUND: Normal-tension glaucoma (NTG) that occurs despite normal intraocular pressure has genetic predisposition. Since retinal ganglion cells (RGCs) are a key node in pathogenesis of glaucoma, neurodegeneration of RGCs is thought to be the main cause increasing the risk of NTG development. Here, we aimed to investigate the association of polymorphisms in RGC development genes with NTG development. MATERIALS AND METHODS: We performed a case-control association study of 435 patients with NTG and 419 normal controls. We genotyped four single nucleotide polymorphisms (SNPs) in genes responsible for RGC development, namely POU4F2 (rs13152799 and rs1504360), POU4F1 (rs9601092), and ISL1 (rs2288468), by either real-time PCR or PCR-RFLP, and evaluated its association with the risk of NTG development. RESULTS: No significant association was observed between the candidate SNPs and NTG development. CONCLUSIONS: To the best of our knowledge, this is the first report exploring the association between genes regulating RGC development and NTG susceptibility. Our data could provide a reference for further researches that focus on finding additional potential SNPs of POU4F2, POU4F1, ISL1 or other RGC development genes for NTG.

Progesterone Receptor Expression in the Benign Prostatic Hyperplasia and Prostate Cancer Tissues, Relation with Transcription, Growth Factors, Hormone Reception and Components of the AKT/mTOR Signaling Pathway.

BACKGROUND: Progesterone receptor (PR) is a critical regulator in reproductive tissues that controls a variety of cellular processes. The objective of the study was to study the PR expression in patients with benign prostatic hyperplasia and prostate cancers in connection with the transcription, growth factors, AR, ERα, ERß, and components of the AKT/mTOR signaling pathway expression. MATERIALS AND METHODS: Ninety-seven patients with prostate pathology were enrolled in the study. Forty-two patients had benign prostatic hyperplasia (BH). Fifty-five patients had locally advanced prostate cancer (PCa). The PSA level and the amount of testosterone in the serum were measured using an ELISA assay. The expression level of NF-κB p65, NF-κB p50, HIF-1, HIF-2, growth factor VEGF, VEGFR2, CAIX, as well as AR, ERα, ERß, PR, Brn-3α, TRIM16 were quantified by RT-PCR. The protein level of Brn-3α, TRIM16 was detected by Western Blotting. RESULTS: Growth in PR expression was observed in PCa tissues compared to BH ones without changes in the clinical and pathological features of the patients. An increase in PR expression was detected in patients with PCa compared to BH. Its mRNA level depended on the expression of AR, Brn-3α, and TRIM16, components of the AKT/mTOR signaling pathway, transcription, and growth factors. An increase in the TRIM16 expression in the PCa tissues was noted in the case of a low PR level. We revealed the growth in PR expression was accompanied by the suppression of the signaling cascade activity, AR, Brn-3α mRNA level, and the enhanced PTEN expression in PCa tissues. The increase in PR expression in PCa led to a decrease in the level of mRNA of NF-κB, HIF-1, VEGF, and VEGFR2. CONCLUSION: In general, the data indicated the significance of the PR expression in the development of the prostate pathology that affected the cross-talk between the steroid hormone reception and signal transduction. 
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Transplantation of Retinal Progenitor Cells from Optic Cup-Like Structures Differentiated from Human Embryonic Stem Cells In Vitro and In Vivo Generation of Retinal Ganglion-Like Cells.

Human embryonic stem cells (hESCs) have the potential to differentiate along the retinal lineage. We have efficiently differentiated human pluripotent stem cells into optic cup-like structures by using a novel retinal differentiation medium (RDM). The purpose of this study was to determine whether the retinal progenitor cells (RPCs) derived from hESCs can integrate into the host retina and differentiate into retinal ganglion cells (RGCs) in vivo. In this study, hESCs (H9-GFP) were induced to differentiate into optic cup-like structures by using our novel differentiation system. The RPCs extracted from the optic cup-like structures were transplanted into the vitreous cavity of N-methyl-d-aspartic acid-treated mice. Sham-treated eyes received the same amount of RDM. The host retinas were analyzed by triple immunofluorescence on the fourth and fifth weeks after transplantation. The optic cup-like structures were efficiently differentiated from hESCs by using our novel differentiation system in vitro for 6-8 weeks. The RPCs extracted from the optic cup-like structures migrated and integrated into the ganglion cell layer (GCL) of the host retina. Furthermore, the remaining transplanted cells were spread over the GCL and had a complementary distribution with host residual RGCs in the GCL of the mouse retina. Surprisingly, some of the transplanted cells expressed the RGC-specific marker Brn3a. These findings demonstrated that the RPCs derived from hESCs could integrate into the host GCL and differentiate into retinal ganglion-like cells in vivo, suggesting that RPCs can be used as an ideal source in supplying countless RGC and embryonic stem cell-based replacement therapies may be a promising treatment to restore vision in patients with degenerative retinal diseases.

Reception of Sex Steroid Hormones in Thyroid Papillary Cancer Tissue and Relationship with Expression and Content of Transcription Factors Brn-3α and TRIM16.

We studied reception of sex steroid hormones in the tissues of thyroid papillary cancer and benign tumor. Enhanced expression of AR and ERß mRNA reflected malignant tumor growth. Nuclear factors Brn-3α and TRIM16 modulating expression of steroid hormones play an important role in the development of thyroid tumors. It was found that the level of TRIM16 mRNA is associated with the expression of ERß, which seems to be mediated by its antiestrogen effect.

Microglia mediate non-cell-autonomous cell death of retinal ganglion cells.

Excitotoxicity is well known in the neuronal death in the brain and is also linked to neuronal damages in the retina. Recent accumulating evidence show that microglia greatly affect excitotoxicity in the brain, but their roles in retina have received only limited attention. Here, we report that retinal excitotoxicity is mediated by microglia. To this end, we employed three discrete methods, that is, pharmacological inhibition of microglia by minocycline, pharmacological ablation by an antagonist for colony stimulating factor 1 receptor (PLX5622), and genetic ablation of microglia using Iba1-tTA::DTAtetO/tetO mice. Intravitreal injection of NMDA increased the number of apoptotic retinal ganglion cells (RGCs) followed by reduction in the number of RGCs. Although microglia did not respond to NMDA directly, they became reactive earlier than RGC damages. Inhibition or ablation of microglia protected RGCs against NMDA. We found up-regulation of proinflammatory cytokine genes including Il1b, Il6 and Tnfa, among which Tnfa was selectively blocked by minocycline. PLX5622 also suppressed Tnfa expression. Tumor necrosis factor α (TNFα) signals were restricted in microglia at very early followed by spreading into other cell types. TNFα up-regulation in microglia and other cells were significantly attenuated by minocycline and PLX5622, suggesting a central role of microglia for TNFα induction. Both inhibition of TNFα and knockdown of TNF receptor type 1 by siRNA protected RGCs against NMDA. Taken together, our data demonstrate that a phenotypic change of microglia into a neurotoxic one is a critical event for the NMDA-induced degeneration of RGCs, suggesting an importance of non-cell-autonomous mechanism in the retinal neuronal excitotoxicity.

Postnatal developmental dynamics of cell type specification genes in Brn3a/Pou4f1 Retinal Ganglion Cells.

BACKGROUND: About 20-30 distinct Retinal Ganglion Cell (RGC) types transmit visual information from the retina to the brain. The developmental mechanisms by which RGCs are specified are still largely unknown. Brn3a is a member of the Brn3/Pou4f transcription factor family, which contains key regulators of RGC postmitotic specification. In particular, Brn3a ablation results in the loss of RGCs with small, thick and dense dendritic arbors ('midget-like' RGCs), and morphological changes in other RGC subpopulations. To identify downstream molecular mechanisms underlying Brn3a effects on RGC numbers and morphology, our group recently performed a RNA deep sequencing screen for Brn3a transcriptional targets in mouse RGCs and identified 180 candidate transcripts. METHODS: We now focus on a subset of 28 candidate genes encoding potential cell type determinant proteins. We validate and further define their retinal expression profile at five postnatal developmental time points between birth and adult stage, using in situ hybridization (ISH), RT-PCR and fluorescent immunodetection (IIF). RESULTS: We find that a majority of candidate genes are enriched in the ganglion cell layer during early stages of postnatal development, but dynamically change their expression profile. We also document transcript-specific expression differences for two example candidates, using RT-PCR and ISH. Brn3a dependency could be confirmed by ISH and IIF only for a fraction of our candidates. CONCLUSIONS: Amongst our candidate Brn3a target genes, a majority demonstrated ganglion cell layer specificity, however only around two thirds showed Brn3a dependency. Some were previously implicated in RGC type specification, while others have known physiological functions in RGCs. Only three genes were found to be consistently regulated by Brn3a throughout postnatal retina development - Mapk10, Tusc5 and Cdh4.

Subtle thinning of retinal layers without overt vascular and inflammatory alterations in a rat model of prediabetes.

Purpose: Diabetic retinopathy is a neurovascular disease characterized by increased permeability of the blood-retinal barrier, changes in the neural components of the retina, and low-grade chronic inflammation. Diabetic retinopathy is a major complication of diabetes; however, the impact of a prediabetic state on the retina remains to be elucidated. The aim of this study was to assess possible early retinal changes in prediabetic rats, by evaluating changes in the integrity of the blood-retinal barrier, the retinal structure, neural markers, and inflammatory mediators. Methods: Several parameters were analyzed in the retinas of Wistar rats that drank high sucrose (HSu; 35% sucrose solution during 9 weeks, the prediabetic animal model) and were compared with those of age-matched controls. The permeability of the blood-retinal barrier was assessed with the Evans blue assay, and the content of the tight junction proteins and neural markers with western blotting. Optical coherence tomography was used to evaluate retinal thickness. Cell loss at the ganglion cell layer was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay and by evaluating the immunoreactivity of the Brn3a transcription factor. To assess retinal neuroinflammation, the mRNA expression and protein levels of inducible nitric oxide synthase isoform (iNOS), interleukin-1 beta (IL-1ß), and tumor necrosis factor (TNF) were evaluated. Iba1 and MHC-II immunoreactivity and translocator protein (TSPO) mRNA levels were assessed to study the microglial number and activation state. Results: The thickness of the inner retinal layers of the HSu-treated animals decreased. Nevertheless, no apoptotic cells were observed, and no changes in retinal neural markers were detected in the retinas of the HSu-treated animals. No changes were detected in the permeability of the blood-retinal barrier, as well as the tight junction protein content between the HSu-treated rats and the controls. In addition, the inflammatory parameters remained unchanged in the retina despite the tendency for an increase in the number of retinal microglial cells. Conclusions: In a prediabetic rat model, the retinal structure is affected by the thinning of the inner layers, without overt vascular and inflammatory alterations. The results suggest neuronal dysfunction (thinning of the inner retina) that may precede or anticipate the vascular and inflammatory changes. Subtle structural changes might be viewed as early disturbances in an evolving disease, suggesting that preventive strategies (such as the modification of diet habits) could be applied at this stage, before the progression toward irreversible dysfunction and damage to the retina.