Progress on spermatogonial stem cell microenvironment.

Spermatogonial stem cells (SSCs) are germ cells (GCs) with long-term self-renewal and differentiation potential in testis, namely tissue stem cells located on the basement membrane, whose self-renewal and differentiation are regulated by the surrounding microenvironment. In recent years, the research of SSCs has made a series of important progress, which brings the hope for the clinical treatment of some male infertility patients. Among them, the microenvironment is particularly important in regulating SSCs. The microenvironment is responsible for integrating the effects of different types of cell components, extracellular matrix, extracellular regulatory molecules and hormones on SSCs, thus regulating the fate of SSCs. The research on SSCs microenvironment has gradually become one of the main contents of stem cell research. In this review, we mainly summarize the cell composition, regulatory factors and characteristics of mouse SSCs microenvironment, thereby providing background information for in-depth study on the structure and function of SSCs microenvironment, and opportunity to find more abundant cell phenotypes and microenvironmental factors through multiple research models in the future.

Lrrc34 Is Highly Expressed in SSCs and Is Necessary for SSC Expansion In Vitro.

Objective To discover critical genes contributing to the stemness and maintenance of spermatogonial stem cells (SSCs) and provide new insights into the function of the leucine-rich repeat (LRR) family member Lrrc34 (leucine-rich repeat-containing 34) in SSCs from mice. Methods Bioinformatic methods, including differentially expressed gene (DEG), gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, were used to uncover latent pluripotency-related genes. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence analyses were utilized to verify the mRNA and protein expression levels, respectively. RNA interference of Lrrc34 using siRNA was performed to detect its transient impact on SSCs. Results Eight DEGs between ID4-EGFP+ (G) and ID4-EGFP+/TSPAN8High (TH), eight DEGs between G and ID4-EGFP+/TSPAN8Low (TL) and eleven DEGs between TH and TL were discovered, and eleven protein-protein interaction (PPI) modules were found to be significant in the PPI network of DEGs. One of the DEGs, Lrrc34, was selected as a potential pluripotency-related gene due to its differential expression among ID4-EGFP+ spermatogonia subsets and its interaction with fibroblast growth factor 2 in the fifth module. Immunofluorescence experiments exhibited specific expression of Lrrc34 in a subpopulation of undifferentiated spermatogonia marked by LIN28A, and RT-PCR experiments confirmed the high expression of Lrrc34 in SSCs from P7 and adult mice. The transient knockdown of Lrrc34 in SSCs resulted in reduced colony sizes and significant changes in the transcriptome and apoptotic pathways. Conclusion Lrrc34 is highly expressed in mouse SSCs and is required for SSC proliferation in vitro through effects on transcriptome and signaling transduction pathways.

Adipocytes promote nicotine-induced injury of endothelial cells via the NF-κB pathway.

Cigarette smoking is one of the most important risk factors of atherosclerosis, which can induce endothelial injury. Meanwhile, adipocytes are the main cell type of perivascular adipose tissue (PVAT), the largest endocrine and paracrine organ and direct anatomical connection to adventitia, which may play a key role in the injury of endothelial cells. We used nicotine to induce dysfunctional HUVECs and adipocytes. In addition, we used a novel model to co-culture HUVECs and adipocytes in vitro by the transwell co-culture system to determine the effect of adipocytes on endothelial injury. Cell apoptosis was detected by Annexin V-FITC. Genes and proteins involved in the nuclear factor kappa B (NF-κB) signaling pathway were detected by qRT-PCR and western blot analysis, respectively. We also investigated the nuclear translocation of NF-κB p65 using immunofluorescence staining. Our results showed that nicotine dose-dependently induces the apoptosis of HUVECs and adipocytes and is associated with increased IKKß and NF-κB p65 expression and with IkBα degradation. Meanwhile through the co-culture system, adipocytes promoted the expression of IKKß and NF-κB p65, as well as the translocation of NF-κB p65, and they accelerated the degradation of IkBα, resulting in increased apoptosis of HUVECs compared with that of the single cultured system. In conclusion, adipocytes could promote endothelial injury via the NF-κB pathway. Moreover, the NF-κB pathway plays pivotal roles in nicotine-induced vascular injury.

Unexpected Neuroprotective Effects of Loganin on 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Neurotoxicity and Cell Death in Zebrafish.

1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), which induces the pathological characteristics of Parkinson's disease in rodents, also specifically targets dopaminergic neurons in zebrafish embryos and larvae. Loganin, a traditional Chinese drug, was reported to regulate immune function and possess anti-inflammatory and anti-shock effects. Here, we investigate the role of loganin in MPTP-induced Parkinson-like abnormalities in zebrafish. MPTP treatment-induced abnormal development, in larvae, such as pericardium edema, increased yolk color, yolk sac edema, and retarded yolk sac resorption, as well as defects in brain development. Loganin could block MPTP-induced defects, with little toxicity to the eggs. Results of whole mount in situ hybridization showed loganin prevented the loss of both dopaminergic neurons and locomotor activity, exhibited by larvae treated with MPTP. In addition, loganin significantly rescued MPTP-induced neurotoxicity on PC12 cells, possibly through the suppression of PI3K/Akt/mTOR axis and JNK signaling pathways. In conclusion, loganin blocks MPTP-induced neurotoxicity and abnormal development in zebrafish. J. Cell. Biochem. 118: 615-628, 2017. © 2016 Wiley Periodicals, Inc.

Activating transcription factor 3 promotes spinal cord regeneration of adult zebrafish.

Zebrafish is an excellent model to study the mechanisms underlying successful central nervous system (CNS) regeneration. Previous study shows that activating transcription factor 3 (ATF3) promotes neurite outgrowth and is involved in optic nerve regeneration in zebrafish. Here, we used zebrafish model to investigate the role of ATF3 in regeneration following spinal cord injury (SCI). Quantitative polymerase chain reaction (qPCR) and in situ hybridization revealed that ATF3 mRNA levels increased at 12 h and 6 d following SCI. Double labeled immunofluorescence showed that ATF3 expressed in motoneurons. Treatment of anti-sense ATF3 morpholino (MO) inhibited locomotor recovery and decreased axon regeneration of spinal cord injured zebrafish. Further, inhibition of ATF3 up-regulated the expression of inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). These data suggest that ATF3 could promote locomotor recovery and axon regrowth in zebrafish SCI model possibly by regulating inflammatory response.

Intradiencephalon injection of histamine inhibited the recovery of locomotor function of spinal cord injured zebrafish.

Human spinal cord injury (SCI) usually causes irreversible disability beneath the injured site due to poor neural regeneration. On the contrary, zebrafish show significant regenerative ability after SCI, thus is usually worked as an animal model for studying neuroregeneration. Most of the previous SCI studies focused on the local site of SCI, the supraspinal-derived signals were rarely mentioned. Here we showed that intradiencephalon injection of histamine (HA) inhibited the locomotor recovery in adult zebrafish post-SCI. Immunofluorescence results showed that intradiencephalon HA administration increased the activated microglia 3 days post injury (dpi), promoted the proliferation of radial glial cells at 7 dpi and affected the morphology of radial glial cells at 11 dpi. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) results showed that intradiencephalon HA administration also reduced the expression of neurotrophic factors including brain-derived neurotrophic factor (BDNF) and insulin-like growth factor1 (IGF-1) at the lesion site, however, had no effect on the expression of pro-inflammatory factors such as TNF-alpha and IL-1 beta. Hence, our data suggested that exogenous intradiencephalon HA retarded locomotor recovery in spinal cord injured zebrafish via modulating the repair microenvironment.

[Expression Patterns of the Proteins Associated with Cell Junctions in Mouse Testes].

OBJECTIVE: To study on the expression patterns of proteins associated with cell junctions in the developing mouse testes. METHOD: The expression levels of reproductive related cell lines spermatogonia cell line GC1 spg, spermatocyte cell line GC2 spg, leydig cell line TM3, and sertoli cell line TM4, primary sertoli cells, and 1-6-week mouse testes were analyzed using Western blot. RESULTS: The sertoli cell junction-associated membrane proteins adhesion molecule A, Occludin and Claudin, and the sertoli-germ cell junction-associated membrane proteins junctional adhesion molecule C, Nectin-3, and E-cadherin were stage-specific in the seminiferous tubules in the mouse testes. The adaptor proteins associated with cell juctions zonula occludens-1, zonula occludens-2, Afadin, Β-catenin, and CD2-associated protein were not stage-specific in the seminiferous tubules in the mouse testes. CONCLUSIONS: In the seminiferous tubules in the mouse testes, the membrane proteins associated with cell junctions are stage-specific. However, the expressions of adaptor proteins associated with cell junctions do not obviously change.

Protective effect of melatonin on cigarette smoke-induced restenosis in rat carotid arteries after balloon injury.

Vascular restenosis after the interventional angioplasty remains the main obstacle to a favorable long-term patency. Many researches suggest cigarette smoking is one of the most important causes of restenosis. This study was designed to investigate whether melatonin could protect against the cigarette smoke-induced restenosis in rat carotid arteries after balloon injury. Three groups of male rats (normal condition, cigarette smoke exposed, cigarette smoke exposed, and melatonin injected) were used in this study. An established balloon-induced carotid artery injury was performed, and the carotid arteries were harvested from these three groups 14 days later. The ratio of intima to media, the infiltration of inflammatory cells, the expression of inflammatory cytokines (NF-κB, IL-1ß, IL-6, TNF-α, MCP-1), adhesion molecules (ICAM-1, VCAM-1), and eNOS were measured. The results showed that cigarette smoke exposure aggravated the stenosis of the lumen, promoted the infiltration of inflammatory cells and induced the expression of the inflammatory cytokines and adhesion molecules after the balloon-induced carotid artery injury. Moreover, cigarette smoke exposure can inhibit the expression of eNOS. Particularly, we surprised that melatonin could minimize this effect caused by cigarette smoke. These results suggested that melatonin could prevent the cigarette smoke-induced restenosis in rat carotid arteries after balloon injury and the mechanism of its protective effect may be the inhibition of the inflammatory reaction. This also implies melatonin has the potential therapeutic applicability in prevention of restenosis after the vascular angioplasty in smokers.

[Identification of proteins interacted with Bat3 using tandem affinity purification].

OBJECTIVE: To identify the specific protein interactions involved in Bat3-mediated apoptosis. METHODS: Tandem affinity purification (TAP) was utilized to investigate Bat3-protein interactions, during which full-length human Bat3 fused with Strep2 and FLAG tag as a bait was used to screen the specific protein-protein interactions. The isolated proteins were identified with mass spectrometry. RESULTS: TAP studies showed that Ubl4A was identified as a Bat3-binding partner. Further investigation using co-immunoprecipitation confirmed that Bat3 was associated with Ubl4A. CONCLUSION: TAP was successfully established and is suitable for isolating the binding partners of Bat3.

Long-term hypoxic hUCMSCs-derived extracellular vesicles alleviates allergic rhinitis through triggering immunotolerance of their VEGF-mediated inhibition of dendritic cells maturation.

BACKGROUND: Extensions of mesenchymal stem cells (MSCs) in vitro may lead to the loss of their biological functions. However, hypoxic culturation has been shown to enhance the proliferation, survival, and immunomodulatory capacity of MSCs. OBJECTIVE: We aimed to investigate the effects of long-term hypoxic cultivation on the properties of human umbilical cord-derived MSCs (hUCMSCs) and the therapeutic effects of their extracellular vesicles (EVs) in allergic rhinitis (AR). METHODS: Proliferation, senescence, telomerase activity and multipotent properties of hUCMSCs were analyzed under long-term culturation of hypoxia (1%) or normoxia (21%), and the therapeutic effects of their conditional medium (CM) and EVs were evaluated in OVA-induced AR mice. Effects of hypoxia-EVs (Hy-EVs) or normoxia-EVs (No-EVs) on human monocyte-derived dendritic cells (DCs) were investigated, and the possible mechanisms of Hy-EVs in induction of immunotolerance were further explored. RESULTS: Long-term hypoxia significantly promoted the proliferation, inhibited cell senescence, maintained the multipotent status of hUCMSCs. Hy-CM and Hy-EVs showed better therapeutic effects in AR mice compared to No-EVs, seen as improvement of AR-related behaviors such as rubbing and sneezing, and attenuation of inflammation in nasal tissues. In addition, Hy-EVs significantly reduced the expressions of HLA-DR, CD80, CD40, and CD83 induced by OVA plus LPS in DCs, inhibiting the maturation of DCs. Furthermore, we observed that VEGF was remarkably enriched in Hy-EVs, but not in No-EVs, and the inhibition of DCs maturation was markedly neutralized by VEGF antibodies, suggesting that VEGF derived from Hy-EVs was responsible for the inhibition of DCs maturation. CONCLUSION: Our results demonstrated that long-term hypoxia significantly promoted the proliferation, inhibited cell senescence, maintained the multipotent status of hUCMSCs, and hypoxia treated hUCMSCs-derived EVs enhanced their therapeutic effects in AR mice through VEGF-mediated inhibition of DCs maturation.

Synergistic effects of combination treatment with bortezomib and doxorubicin in human neuroblastoma cell lines.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor in infants. Currently, the mainstay of NB chemotherapy is combination treatment with some traditional drugs, but these combination regimens are always inefficient. METHODS: The aim of this study was to evaluate the inhibitory effect of a combination of doxorubicin and bortezomib, a novel anticancer drug and the first prote-asome inhibitor approved for the treatment of human malignant tumors, on the proliferation of two human NB cell lines, SK-N-SH and SH-SY5Y. The general mechanism underlying this combined effect was also investigated. Synergistic inhibitory effects on human NB cell proliferation were evaluated using the median-effect principle. The pro-apoptotic effects of these drugs were evaluated using double staining with annexin-V-FITC and propidium iodide. RESULTS: Synergistic inhibitory effects on proliferation were observed when a combination of bortezomib and doxorubicin was applied to cultured NB cells. A similar synergistic effect on apoptosis was also observed when the two drugs were used concurrently, which suggested that the possible mechanism underlying the observed synergistic inhibitory effect might be related to apoptosis. CONCLUSION: The combination of bortezomib and doxorubicin appears to be a promising strategy to treat NB.

[Construction and identification of nemo-like kinase gene recombinant adenovirus vector].

OBJECTIVE: To construct the nemo-like kinase (NLK) gene recombinant adenovirus vector. METHODS: The AdEasy system was used to construct the recombinant adenovirus vector. Using reverse transcriptase polymerase chain reaction (RT-PCR), the full-length gene of NLK and its mutants (K155M, T286V, and C425Y) were amplified from HEK293 cells. The FLAG tag was appended at the C-terminal of NLK. After ligation and transformation, the NLK gene and its mutants were cloned into the pAdTrack-CMV vector. It was detected by PCR, sequencing, and Western blot analysis. Using DNA recombination and homogenous recombination, the normally expressed plasmids were linearized by the restriction enzyme-PmeI and PacI, then the enzyme-digested products were recycled by using ethanol precipitation. The purified product was transfected to HEK293A packaging cells with FuGENE HD transfection reagent. After amplification of the recombinant adenovirus, Western blot analysis was performed to detect the expression of NLK gene and its mutants. RESULTS: The successful construction of pAdtrack-CMV-NLK (and mutants) was confirmed by PCR and sequencing. Western blot analysis showed that the target genes and the recombinant adenovirus were obtained. This recombinant virus was able to express NLK protein and its mutants correctly in HCT 116 cells. CONCLUSION: The NLK gene recombinant adenovirus vector was successfully constructed and identified.

[Establishment of a targeting protein for Xenopus kinesin-like protein 2 C' terminal SBP-3 x Flag tagged HCT 116 colorectal cancer cell model].

OBJECTIVE: To develop a targeting protein for Xenopus kinesin-like protein 2 (TPX2) C' terminal SBP-3 x Flag-tagged HCT 116 cell model. METHODS: Homologous arms were amplified by polymerase chain reaction (PCR), and then the adeno-associated virus (AAV) -targeting vector of TPX2 was constructed. HCT 116 cells were targeted after the viruses were packaged. Positive cell clones with neomycin resistance gene were obtained by G418 and PCR screening. Finally, the neomycin gene cassette was excised after the targeted clones were infected with adenovirus expressing Cre-recombinase, and the TPX2 C' terminal SBP and 3 x Flag endogenous double-tagged HCT 116 cells were obtained by PCR screening. RESULTS: Two positive cell clones with neomycin resistance gene were obtained by PCR screening. The positive clones with neomycin resistance gene excised were obtained by Cre adenovirus infection, and the knock-in of SBP-3 x Flag gene was verified by Western blot analysis. CONCLUSION: The TPX2 C' terminal SBP-3 x Flag tagged HCT 116 cell model was successfully established.

Association of Plasma Connective Tissue Growth Factor Levels with Hyperthyroid Heart Disease.

Hyperthyroid heart disease (HHD) is one of the most severe complications of overt hyperthyroidism and increases the risk of mortality in affected patients. Early identification of patients at a higher risk of developing HHD can improve clinical outcomes through active surveillance and management. Connective tissue growth factor (CTGF), a secreted extracellular protein, plays a significant role in cardiac remodeling and dysfunction. We aimed to investigate the association between plasma CTGF level and the risk of HHD in this study. A total of 142 overt hyperthyroid patients without HHD and 99 patients with HHD were included. The plasma CTGF levels were measured using ELISA kits. Routine clinical medical data and echocardiography parameters were recorded for analysis. The plasma CTGF level was significantly higher in patients with HHD than in those without HHD (P=0.002). The plasma CTGF level was positively correlated with free triiodothyronin, tryrotropin receptor antibody, troponin I and lactate dehydrogenase levels and the left atrium diameters, right atrium diameters, and right ventricular end-diastolic diameters (all P<0.05). Logistic regression analysis showed that quartiles 3 and 4 of plasma CTGF levels were significantly associated with the increased risk of HHD (crude OR: 2.529; 95% CI: 1.188-5.387). However, after adjustment for the potentially confounding variables, quartile 4 alone was significantly associated with the higher risk of HHD relative to quartile 1. Hyperthyroid patients with HHD display higher plasma CTGF levels. Furthermore, CTGF is an independent risk factor for HHD. Therefore, the plasma CTGF level may be a potential biomarker for the risk of HHD.

Adequate expression of neuropeptide Y is essential for the recovery of zebrafish motor function following spinal cord injury.

In strong contrast to limited repair within the mammalian central nervous system, the spinal cord of adult zebrafish is capable of almost complete recovery following injury. Understanding the mechanism underlying neural repair and functional recovery in zebrafish may lead to innovative therapies for human spinal cord injury (SCI). Since neuropeptide Y (NPY) plays a protective role in the pathogenesis of several neurological diseases, in the present study, we evaluated the effects of NPY on neuronal repair and subsequent recovery of motor function in adult zebrafish following SCI. Real-time quantitative PCR (qRT-PCR), in situ hybridization and immunostaining for NPY revealed decreased NPY expression at 12 hours (h), 6 and 21 days (d) after SCI. Double-immunostaining for NPY and islet-1, a motoneuron marker, showed that NPY was expressed in spinal cord motoneurons. Morpholino (MO) treatment for suppressing the expression of NPY inhibited supraspinal axon regrowth and locomotor recovery, in which double-staining for proliferating cell nuclear antigen (PCNA) and islet-1 showed a reduction in motoneuron proliferation. Similarly, a downregulated mRNA level of Y1 receptor of NPY (NPY1R) was also detected at 12 h, 6 and 21 d after injury. Immunostaining for NPY and in situ hybridization for NPY1R revealed that NPY1R was co-localized with NPY. Collectively, the results suggest that NPY expression in motoneurons promotes descending axon regeneration and locomotor recovery in adult zebrafish after SCI, possibly by regulating motoneuron proliferation through activation of NPY1R.

Fruquintinib with gefitinib as first-line therapy in patients carrying EGFR mutations with advanced non-small cell lung cancer: a single-arm, phase II study.

BACKGROUND: Fruquintinib is an oral vascular endothelial growth factor receptor inhibitor. Previous gefitinib studies with anti-angiogenics show promising efficacy. This phase II trial assessed efficacy and safety of fruquintinib in combination with gefitinib, in patients with advanced non-small cell lung cancer (NSCLC). METHODS: Fifty patients with stage IIIB/IV NSCLC and an epidermal growth factor receptor (EGFR) exon-19 deletion or exon-21 L858R mutation were enrolled between January 2017 and June 2019. Per protocol (version 1.0), patients received 4 mg fruquintinib once daily (qd) Days 1-21 of Cycle 1, using a 3-week-on/1-week-off schedule, plus continuous gefitinib 250 mg qd. If tolerated, patients proceeded to fruquintinib 5 mg qd (fruquintinib 5 mg group, n=26). Following protocol updates, dose escalation of fruquintinib from 4 mg qd to 5 mg qd was not allowed. The primary efficacy endpoint was objective response rate (ORR); secondary endpoints included progression-free survival (PFS), disease control rate (DCR), time to response, duration of response and adverse events (AEs). RESULTS: ORR was 73.5% (95% CI, 58.9-85.1) and DCR was 98.0% (95% CI, 89.2-100.0). Median PFS was 14.7 months for both groups; PFS was highest for patients with exon-19 deletion (16.5 months; 95% CI, 12.9-21.2). Grade ≥3 treatment-emergent AEs occurred in 17 (65.3%; fruquintinib 5 mg,) and 11 patients (45.8%; 4 mg). Serious AEs were recorded for nine patients (fruquintinib 5 mg, six patients; 4 mg, three). CONCLUSIONS: Fruquintinib and gefitinib treatment showed an acceptable safety profile and promising efficacy in patients with NSCLC.

Regulation of the G2/M phase of the cell cycle by sperm associated antigen 8 (SPAG8) protein.

Sperm associated antigen 8 (SPAG8), a testis-specific protein produced during male germ cell differentiation, was isolated from a human testis expression library using antibodies found in the serum obtained from an infertile woman. It was found to have a close functional relationship with microtubules. In this study, we generated a stably expressing SPAG8 CHO-K1 cell line. Immunofluorescence confocal microscopy showed that SPAG8 was concentrated at the microtubule-organizing center (MTOC) during prophase. As the cells progressed into metaphase, it co-localized with alpha-tubulin on the spindle. In anaphase, it was detected on both astral microtubules and mid-zone. Following cytokinesis, SPAG8 resumed its localization on the MTOC. Meanwhile, flow cytometry analysis found that SPAG8 prolonged the G2/M phase of CHO-K1 cells stably expressing SPAG8. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that SPAG8 inhibited the proliferation of the stable cells. SPAG8 might be involved in the regulation of cell cycle by changing the phosphorylation level of Tyr15 on cdc2. These results suggest that SPAG8 might play a role in cell division during spermatogenesis.

[Expression, purification, and crystallization of a novel galactose mutarotase from Thermoanaerobacter tengcongensis].

OBJECTIVE: To purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction. METHODS: The tte 1925 gene was subcloned into the prokaryotic expression vector pGEX-6P-1 and overexpression was obtained in the E.coli BL21 (DE3) through transformation of the right recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with glutathione S-transferase tag expressed highly by the induction of isopropyl beta-D-thiogalactoside and was purified in a three-step procedure, which included Glutathione Sepharose 4B affinity, ion chromatography (Resource Q 6 mL), and gel filtration chromatography (10/300 superdex 200). RESULT: The purity of the purified protein was over 99% and a large amount of claval crystals whose X-ray diffraction reached 1.9 A were obtained. CONCLUSIONS: We successfully prepared TTE1925 with high purity and obtained crystals for X-ray diffraction. These work paved the way for the further research on the 3-D structure of TTE1925 and its biological mechanism.

Modulation of transcriptional corepressor activity of prospero-related homeobox protein (Prox1) by SUMO modification.

Prospero-related homeobox protein (Prox1) plays essential roles in the development of many tissues and organs. In the present study, we show that Prox1 is modified by the small ubiquitin-like protein SUMO-1 in cultured cells. Mutation analysis identified at least four potential sumoylation sites within the repression domain of Prox1. Our data indicate that sumoylation of Prox1 reduces its interaction with HDAC3 and as a result downregulates its corepressor activity. These findings suggest that sumoylation may serve as a novel mechanism for the regulation of Prox1's corepressor activity.