Prox1 Expression Research Articles

Reduced Expression of PROX1 Transitions Glioblastoma Cells into a Mesenchymal Gene Expression Subtype.

The homeodomain transcription factor PROX1 has been linked to several cancer types, including gliomas, but its functions remain to be further elucidated. Here we describe a functional role and the prognostic value of PROX1 in glioblastoma. Low expression of PROX1 correlated with poor overall survival and the mesenchymal glioblastoma subtype signature. The latter finding was recapitulated in vitro, where suppression or overexpression of PROX1 in glioma cell cultures transitioned cells to a mesenchymal or to a nonmesenchymal glioblastoma gene expression signature, respectively. PROX1 modulation affected proliferation rates that coincided with changes in protein levels of CCNA1 and CCNE1 as well as the cyclin inhibitors CDKN1A, CDKN1B, and CDKN1C. Overexpression of SOX2 increased PROX1 expression, but treatment with a CDK2 inhibitor subsequently decreased PROX1 expression, which was paralleled by decreased SOX2 levels. The THRAP3 protein was a novel binding partner for PROX1, and suppression of THRAP3 increased both transcript and protein levels of PROX1. Together, these findings highlight the prognostic value of PROX1 and its role as a regulator of glioblastoma gene expression subtypes, intratumoral heterogeneity, proliferation, and cell-cycle control.Significance: These findings demonstrate the role and prognostic value of PROX1 in glioblastomas; low PROX1 levels correlate with a mesenchymal gene expression subtype and shorter survival in glioblastoma tumors. Cancer Res; 78(20); 5901-16. ©2018 AACR.

Transcription Factor PROX1 Suppresses Notch Pathway Activation via the Nucleosome Remodeling and Deacetylase Complex in Colorectal Cancer Stem-like Cells.

The homeobox transcription factor PROX1 is induced by high Wnt/β-catenin activity in intestinal adenomas and colorectal cancer, where it promotes tumor progression. Here we report that in LGR5+ colorectal cancer cells, PROX1 suppresses the Notch pathway, which is essential for cell fate in intestinal stem cells. Pharmacologic inhibition of Notch in ex vivo 3D organoid cultures from transgenic mouse intestinal adenoma models increased Prox1 expression and the number of PROX1-positive cells. Notch inhibition led to increased proliferation of the PROX1-positive colorectal cancer cells, but did not affect their ability to give rise to PROX1-negative secretory cells. Conversely, PROX1 deletion increased Notch target gene expression and NOTCH1 promoter activity, indicating reciprocal regulation between PROX1 and the Notch pathway in colorectal cancer. PROX1 interacted with the nucleosome remodeling and deacetylase (NuRD) complex to suppress the Notch pathway. Thus, our data suggests that PROX1 and Notch suppress each other and that PROX1-mediated suppression of Notch mediates its stem cell function in colorectal cancer.Significance: These findings address the role of the PROX1 homeobox factor as a downstream effector of Wnt/β-catenin singling in colorectal cancer stem cells and show that PROX1 inhibits the Notch pathway and helps to enforce the stem cell phenotype and inhibit differentiation. Cancer Res; 78(20); 5820-32. ©2018 AACR.

Abstract 387: Lymphatic Endothelial Cells Derived Exosomes Promote Neolymphangiogenesis After Injury

Neolymphangiogenesis after myocardial infarction (MI) has been extensively documented; however the source of new lymphatic vessels (LVs) is still unknown. It has been described that new LVs may derive from resident lymphatic endothelial cells (LECs) or from trans-differentiation of endothelial cells (ECs) and pericytes from veins into LECs. Which signals activate both the spreading and the trans-differentiation needs to be investigated. LECs are known to release transcytotic vesicles and exosomes, thus, we hypnotized that the enriched protein and RNA cargo of LECs derived exosomes may promote the new LVs formation after injury. Exosomes derived from LECs were isolated from LECs cultured conditioned medium and HUVECs were treated with different concentration of exosomes cargo for 10 days. After treatment, q-PCR for the major endothelial and lymphatic endothelial markers was performed on treated HUVECs. In a dose dependent manner we observed a decreased expression of KDR and CD31, endothelial markers that are not expressed by LECs and an increased expression of Prox-1 and VEGFR3. Prox-1 is the major lymphatic endothelial transcription factor that is not activated in ECs and is responsible for the transcription of LYVE-1 and podoplanin. VEGFR3 is the only known vascular endothelial growth factor receptor expressed on LECs which is not displayed by ECs. Next, we evaluated the effect of LECs derived exosomes on in vitro lymphangiogenesis assay by measuring the ability of LECs to organize into capillary-like structures on matrigel. LECs treated with LECs derived exosomes are able to form tubes and branches as early as within 1 hour compared to untreated cells. Our preliminary results show cargo of exosomes from LECs activates the signal transduction and metabolism of LECs and HUVECs promoting the new LVs formation through spreading and trans-differentiation of the targeted cells.

The transcription factor prospero homeobox protein 1 is a direct target of SoxC proteins during developmental vertebrate neurogenesis.

The high-mobility-group domain containing SoxC transcription factors Sox4 and Sox11 are expressed and required in the vertebrate central nervous system in neuronal precursors and neuroblasts. To identify genes that are widely regulated by SoxC proteins during vertebrate neurogenesis we generated expression profiles from developing mouse brain and chicken neural tube with reduced SoxC expression and found the transcription factor prospero homeobox protein 1 (Prox1) strongly down-regulated under both conditions. This led us to hypothesize that Prox1 expression depends on SoxC proteins in the developing central nervous system of mouse and chicken. By combining luciferase reporter assays and over-expression in the chicken neural tube with invivo and invitro binding studies, we identify the Prox1 gene promoter and two upstream enhancers at -44kb and -40kb relative to the transcription start as regulatory regions that are bound and activated by SoxC proteins. This argues that Prox1 is a direct target gene of SoxC proteins during neurogenesis. Electroporations in the chicken neural tube furthermore show that Prox1 activates a subset of SoxC target genes, whereas it has no effects on others. We propose that the transcriptional control of Prox1 by SoxC proteins may ensure coupling of two types of transcription factors that are both required during early neurogenesis, but have at least in part distinct functions. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Abstract 1143: Prox1 and Notch mark distinct colorectal cancer stem cell populations

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality. Signaling pathways, such as the Wnt and Notch pathways, are essential for the maintenance and differentiation of wild-type intestinal stem cells. We have shown that the Prox1 transcription promotes expansion of the colorectal cancer stem cell pool and that a subpopulation of the Prox1+ CRC cells display stem cell activity. Because of these results we have further analyzed the role of Prox1 in the regulation of CRC stem cell. Here, we report that Prox1 regulates CRC stem-like cells via bidirectional interaction with Notch1 during CRC initiation and progression. Using genetic in vivo models and ex vivo 3D organoid cultures, we show that Notch inhibition decreases the number of Lgr5+ stem cells whereas it increases expression of the Prox1. Although Notch inhibition led to increased proliferation of the Prox1 positive cells, it did not affect their ability to give rise to differentiated Prox1 negative progeny. Ectopic overexpression of the active fragment of Notch1 suppressed Prox1 expression and inhibited stem cell activity in the CRC cells. On the other hand, the PROX1-NuRD complex suppressed the Notch signaling pathway and Prox1 deletion increased Notch target gene expression and promoter activity, indicating reciprocal regulation between Prox1 and Notch1. Thus, although Prox1 and Notch suppress each other in colorectal cancer cells, Prox1+ cells can function as a stem cell population without the need for Notch pathway activity. Citation Format: Jenny Högström, Sarika Heino, Pauliina Kallio, Marianne Lähde, Veli-Matti Leppänen, Seppo Kaijalainen, Diego Balboa, Timo Otonkoski, Sylvie Robine, Zoltan Wiener, Kari Alitalo. Prox1 and Notch mark distinct colorectal cancer stem cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1143.

Decreased Expression of Prox1 Is Associated With Postoperative Recurrence in Crohn's Disease.

Prox1 is a transcription factor necessary for lymphangiogenesis and lymphatic function. The aim of the study was to assess the correlation between the expression of Prox1 and postoperative recurrence in Crohn's disease [CD]. Forty CD patients who underwent ileo-colonic resection were included. Expression levels of Prox1 and D2-40 were detected using immunohistochemistry. Expression levels of Prox1, VEGFR3, and VEGFC protein were also detected in fresh CD specimens using western blotting and quantitative polymerase chain reaction [Q-PCR]. Endoscopic recurrence was used as the endpoint. Patients comprised two groups: endoscopic recurrence [Group R+] and no endoscopic recurrence [Group R-]. Prox1 protein expression was significantly higher in CD than in normal tissues [p <0.05], as detected using both immunohistochemistry and western blotting. Analysis of inter-relationships revealed significant correlation between Prox1 expression and lymphatic vessel density [p <0.001, r = 0.823]. There was also significant correlation between Prox1 expression and the visceral fat area [VFA] [p = 0.002, r = -0.469]. The Group R- patients had significantly higher Prox1 expression than the Group R+ patients [21.08 ± 1.61 versus 15.64 ± 1.17, p = 0.011]. Also, the lymphatic vessel density value was lower in Group R+ than in Group R- patients [6.02 ± 0.39 versus 8.13 ± 0.59, p = 0.004]. Moreover, there was a significant difference in the VFA between Group R- and Group R+ patients [64.43 ± 7.76 versus 90.44 ± 6.11, p = 0.016]. In addition to Prox1, VEGFC/VEGFR3 was found to increase, which was further confirmed using Q-PCR. Prox1 expression could be useful as a protective factor against recurrence in CD patients. The therapeutic role of Prox1 may lead to improved treatments.

Lens-specific deletion of the Msx2 gene increased apoptosis by enhancing the caspase-3/caspase-8 signaling pathway.

ObjectiveTo investigate the influence of Msx2 conditional gene knockout during lens development in mice.MethodsLens-specific Msx2 knockout mice were generated using the Cre-loxP system. The eyes of Msx2 conditional knockout (Msx2CKO) and wild-type (Msx2WT) mice were examined during embryonic and early postnatal periods using histological, immunofluorescence, in situ hybridization, cell proliferation, apoptosis, and mRNA microarray analyses.ResultsMsx2CKO mice exhibited small lens formation and microphthalmia after birth, while Msx2CKO embryos exhibited a persistent lens stalk, small lens formation, and microphthalmia. Conditional deletion of Msx2 also led to an increased apoptosis rate, a significant reduction in FoxE3 expression, and an upregulation of Prox1 expression in the lens vesicle during the early embryonic period. Microarray comparison of Msx2CKO and Msx2WT lens transcriptomes identified a large number of differentially expressed genes. Real-time PCR showed that Casp8 and Casp3 expression was upregulated in Msx2CKO mice at post-natal day 1.ConclusionThe activation of apoptosis through the caspase-8/caspase-3 signaling pathway, together with the downregulation of FoxE3 expression, appeared to account for the smaller lens formation in Msx2CKO mice.

Cooperative Effects of Vascular Angiogenesis and Lymphangiogenesis.

In this study, we modeled lymphangiogenesis and vascular angiogenesis in a microdevice using a tissue engineering approach. Lymphatic vessels (LV) and blood vessels (BV) were fabricated by sacrificial molding with seeding human lymphatic endothelial cells and human umbilical vein endothelial cells into molded microchannels (600 μm diameter). During subsequent perfusion culture, lymphangiogenesis and vascular angiogenesis were induced by addition of phorbol 12-myristate 13-acetate (PMA) and VEGF-C or VEGF-A characterized by podoplanin and Prox-1 expression. The lymphatic capillaries formed button-like junctions treated with dexamethasone. To test the potential for screening anti-angiogenic (vascular and lymphatic) factors, antagonists of VEGF were introduced. We found that an inhibitor of VEGF-R3 did not completely suppress lymphatic angiogenesis with BVs present, although lymphatic angiogenesis was selectively prevented by addition of a VEGF-R3 inhibitor without BVs. To probe the mechanism of action, we focus on matrix metalloproteinase (MMP) secretion by vascular endothelial cells and lymphatic endothelial cells under monoculture or co-culture conditions. We found that vascular angiogenesis facilitated lymphangiogenesis via remodeling of the local microenvironment by the increased secretion of MMP, mainly by endothelial cells. Applications of this model include a drug screening assay for corneal disease and models for tumorigenesis including lymphatic angiogenesis and vascular angiogenesis.

CEACAM1 resists hypoxia-induced inhibition of tube formation of human dermal lymphatic endothelial cells

Tube formation is one of the fundamental events required by angiogenesis and lymphangiogenesis. To date, there is little knowledge on the effects of hypoxia on tube formation of human dermal lymphatic endothelial cells (HDLECs). In this study, we found that tube formation of HDLECs was inhibited under hypoxic condition with decreased expressions of VEGF-D, CEACAM1 and Prox1 genes. However, hypoxia-induced inhibition of tube formation of HDLECs was reversed by conditional media from hypoxic tumor cells. After knockdown of CEACAM1 by siRNA transfection, tube formation of HDLECs was increased with elevated Prox1 expression, suggesting that CEACAM1 downregulates Prox1 and plays an inhibitory role in tube formation of HDLECs. Since the expressions of CEACAM1 and Prox1 were both decreased by hypoxia, there are additional mechanisms downregulating Prox1 expressions during hypoxia-inhibited tube formation of HDLECs.

Tcm Decoction for Restoration Inhibits Hepatic Swelling and Regeneration after Partial Hepatectomy via VEGF-A and PROX1 Downregulation in the Residual Liver Tissue

Objective: To explore the molecular mechanism by which ‘traditional Chinese medicine (TCM) decoction for restoration’ (‘TCM decoction’) affects swelling and pain elimination, by assessing its effects on hepatic swelling and regeneration after partial hepatectomy. Methods: A total of 160 mice were randomized into control and TCM decoction treatment groups. The control animals underwent partial liver resection of left lateral lobe, while the TCM decoction treatment group received oral administration of the decoction by gavage (10 g/kg), twice a day, after the same surgery administered to the control group. Ten mice in each group were sacrificed every other day from postoperative day 1 to 8. VEGF-A and PROX1 protein expression levels were evaluated. Results: Swelling and degeneration of residual liver tissue were significantly alleviated by TCM decoction at the early stage of surgical injury, and subsequent hepatic regeneration was significantly inhibited as well. Interestingly, VEGF-A and PROX1 expression levels in residual liver tissue were also significantly reduced by TCM decoction. Conclusion: Swelling and degeneration of residual liver tissue are significantly alleviated, with hepatic regeneration inhibited by TCM decoction after partial hepatectomy, through VEGF-A and PROX1 downregulation.

Disruption of liver development and coagulation pathway by ochratoxin A in embryonic zebrafish

Ochratoxin A (OTA) is a mycotoxin that is found in various food and feed products. The molecular mechanisms that are associated with OTA hepatotoxicity and teratogenicity have not been extensively elucidated in a developing organism. In this study, the transcriptomic profile of zebrafish embryos indicates that hemostasis and blood coagulation are the top two pathways affected by OTA. The treatment of embryos with OTA was able to decrease the expression of genes that encode coagulation factors and liver markers, including f7, f9b, cp and vtna. OTA also weakened the signal of liver-specific microRNA-122. OTA administration not only reduced the size of a developing embryonic liver, but also decreased the number of phosphorylated histone H3-positive cells by immunohistochemical staining. OTA suppressed the expression of hhex and prox1, two critical transcriptional factors during hepatoblast specification, in the developing liver, but did not alter the insulin signal in the pancreas. In vitro analysis with zebrafish liver (ZFL) cells indicated that OTA blocked the expression of f7, fgb and liver markers. In summary, OTA exposure resulted in the generation of small livers which led to deficiency of coagulation factors in embryonic zebrafish. Impairment of hhex and prox1 gene expression and hepatocyte proliferation contributed to the disruption of liver development mediated by OTA.

The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells.

The prospero homeobox 1 (Prox1) transcription factor is a key player during embryogenesis and lymphangiogenesis. Altered Prox1 expression has been found in a variety of human cancers, including papillary thyroid carcinoma (PTC). Interestingly, Prox1 may exert tumor suppressive or tumor promoting effect, depending on the tissue context. In this study, we have analyzed Prox1 expression in normal and malignant human thyroid carcinoma cell lines. Moreover, we determined the effect of Prox1 silencing and overexpression on the cellular processes associated with the metastatic potential of tumor cells: proliferation, migration, invasion, apoptosis and anchorage-independent growth, in the follicular thyroid carcinoma (FTC) FTC-133 cell line. We found that Prox1 expression was significantly higher in FTC-derived cells than in PTC-derived cells and normal thyroid, and it was associated with the PI3K/Akt signaling pathway. In the FTC-133 cells, it was associated with cell invasive potential, motility and wound closure capacities, but not with proliferation or apoptosis. Modifying Prox1 expression also induced substantial changes in the cytoskeleton structure and cell morphology. In conclusion, we have shown that Prox1 plays an important role in the development of FTC and that its suppression prevents, whereas its overexpression promotes, the malignant behavior of thyroid follicular cancer cells.

High PROX1 expression in gastric cancer predicts better survival.

BackgroundPROX1 is a transcription factor involved in the development of various organs. It has also an important function in colorectal cancer progression. The aim of this study was to investigate the prognostic role of PROX1 expression in gastric cancer.MethodsWe evaluated PROX1 expression in gastric cancer by immunohistochemistry of tumor-tissue microarrays including tumor specimens from 283 patients who underwent surgery at Helsinki University Hospital. We investigated the association of PROX1 expression with clinicopathologic variables and patient survival.ResultsCytoplasmic PROX1 reactivity was high in 56 (20.5%) and low in 217 (79.5%) cases. Low PROX1 immunostaining associated with diffuse cancer type (p = 0.002). In subgroup analysis, PROX1 was a significant marker of better prognosis in patients aged under 66 (p = 0.007), in those with intestinal cancer (p = 0.025), among men (p = 0.019), and in tumors of less than 5 cm diameter (p = 0.030). Patients with high PROX1 expression had a cancer-specific 5-year survival of 65.6% (95% CI 52.7–78.5), compared to 37.1% (95% CI 30.2–44.0) for those with low expression (p = 0.004, log-rank test). This result remained significant in multivariable analysis (HR = 0.56; 95% CI 0.35–0.90; p = 0.017).ConclusionIn gastric cancer, high cytoplasmic PROX1 expression is an independent marker of better prognosis.

Seizure severity-dependent selective vulnerability of the granule cell layer and aberrant neurogenesis in the rat hippocampus.

The pilocarpine-induced status epilepticus rodent model has been commonly used to analyze the mechanisms of human temporal lobe epilepsy. Recent studies using this model have demonstrated that epileptic seizures lead to increased adult neurogenesis of the dentate granule cells, and cause abnormal cellular organization in dentate neuronal circuits. In this study, we examined these structural changes in rats with seizures of varying severity. In rats with frequent severe seizures, we found a clear loss of Prox1 and NeuN expression in the dentate granule cell layer (GCL), which was confined mainly to the suprapyramidal blade of the GCL at the septal and middle regions of the septotemporal axis of the hippocampus. In the damaged suprapyramidal region, the number of immature neurons in the subgranular zone was markedly reduced. In contrast, in rats with less frequent severe seizures, there was almost no loss of Prox1 and NeuN expression, and the number of immature neurons was increased. In rats with no or slight loss of Prox1 expression in the GCL, ectopic immature neurons were detected in the molecular layer of the suprapyramidal blade in addition to the hilus, and formed chainlike aggregated structures along the blood vessels up to the hippocampal fissure, suggesting that newly generated neurons migrate at least partially along blood vessels to the hippocampal fissure. These results suggest that seizures of different severity cause different effects on GCL damage, neurogenesis, and the migration of new neurons, and that these structural changes are selective to subdivisions of the GCL and the septotemporal axis of the hippocampus.

Development and Characterization of A Novel Prox1-EGFP Lymphatic and Schlemm\u2019s Canal Reporter Rat

The lymphatic system plays a key role in tissue fluid homeostasis, immune cell trafficking, and fat absorption. We previously reported a bacterial artificial chromosome (BAC)-based lymphatic reporter mouse, where EGFP is expressed under the regulation of the Prox1 promoter. This reporter line has been widely used to conveniently visualize lymphatic vessels and other Prox1-expressing tissues such as Schlemm’s canal. However, mice have a number of experimental limitations due to small body size. By comparison, laboratory rats are larger in size and more closely model the metabolic, physiological, and surgical aspects of humans. Here, we report development of a novel lymphatic reporter rat using the mouse Prox1-EGFP BAC. Despite the species mismatch, the mouse Prox1-EGFP BAC enabled a reliable expression of EGFP in Prox1-expressing cells of the transgenic rats and allowed a convenient visualization of all lymphatic vessels, including those in the central nervous system, and Schlemm’s canal. To demonstrate the utility of this new reporter rat, we studied the contractile properties and valvular functions of mesenteric lymphatics, developed a surgical model for vascularized lymph node transplantation, and confirmed Prox1 expression in venous valves. Together, Prox1-EGFP rat model will contribute to the advancement of lymphatic research as a valuable experimental resource.

Lymphatic Endothelial Cells under Mechanical Stress: Altered Expression of Inflammatory Cytokines and Fibrosis.

Secondary lymphedema, resulting from damage to lymphatic vessels, is a common sequela following surgical removal of lymph nodes for cancer. Current therapeutics for treating lymphedema are limited and further research on underlying causes is warranted. Published studies on molecular mechanisms of lymphedema primarily focus on lymphatic endothelial cells (LECs), which comprise the innermost lining of lymphatic capillaries and collecting vessels. However, traditional static culture of LECs may not adequately recapitulate the lymphedemous cell phenotype as transcriptomal comparison of human dermal LECs has shown significant differences in ex vivo and in vitro LEC gene expression. In this study, we designed a dynamic culture system, in which LECs were exposed to physiologic and excess mechanical strain to determine if native and lymphedemous phenotypes could be reproduced in vitro. Purified human LECs were cultured in silicon dishes and subjected to 0% (control), 4%, and 8% mechanical strain for 72 hours. Our results indicate that control and stretched LECs maintained a mature phenotype. Extreme stretching at 8% strain significantly increased LEC proliferation and significantly increased Prox1 expression, suggesting a lymphedemous cell phenotype resulting with lymphangiogenesis. Mechanical strain reinforced a mature lymphatic phenotype and excess strain promoted lymphangiogenesis, while altering collagen deposition and cytokine secretion.

Prox1 Is a Marker for AII Amacrine Cells in the Mouse Retina.

The transcription factor Prox1 is expressed in multiple cells in the retina during eye development. This study has focused on neuronal Prox1 expression in the inner nuclear layer (INL) of the adult mouse retina. Prox1 immunostaining was evaluated in vertical retinal sections and whole mount preparations using a specific antibody directed to the C-terminus of Prox1. Strong immunostaining was observed in numerous amacrine cell bodies and in all horizontal cell bodies in the proximal and distal INL, respectively. Some bipolar cells were also weakly immunostained. Prox1-immunoreactive amacrine cells expressed glycine, and they formed 35 ± 3% of all glycinergic amacrine cells. Intracellular Neurobiotin injections into AII amacrine cells showed that all gap junction-coupled AII amacrine cells express Prox1, and no other Prox1-immunostained amacrine cells were in the immediate area surrounding the injected AII amacrine cell. Prox1-immunoreactive amacrine cell bodies were distributed across the retina, with their highest density (3887 ± 160 cells/mm2) in the central retina, 0.5 mm from the optic nerve head, and their lowest density (3133 ± 350 cells/mm2) in the mid-peripheral retina, 2 mm from the optic nerve head. Prox1-immunoreactive amacrine cell bodies comprised ~9.8% of the total amacrine cell population, and they formed a non-random mosaic with a regularity index (RI) of 3.4, similar to AII amacrine cells in the retinas of other mammals. Together, these findings indicate that AII amacrine cells are the predominant and likely only amacrine cell type strongly expressing Prox1 in the adult mouse retina, and establish Prox1 as a marker of AII amacrine cells.

Prox1 represses IL-2 gene expression by interacting with NFAT2.

Interleukin-2 (IL-2) is critical for T lymphocyte activation and regulated by many transcriptional factors. Prospero-related homeobox 1 (Prox1) is a multifunctional transcription factor, which can work as either a transcriptional activator or repressor depending on the cellular and developmental environment. We previously reported the Prox1 expression in T cells, raising the possibility of Prox1 involvement in the regulation of T cell function and IL-2 production. Here we demonstrated that the Prox1 expression in CD4+ T cells was downregulated by T cell receptor (TCR) activation. Overexpression of Prox1 attenuated IL-2 production, while knockdown of endogenous Prox1 by small interfering RNA increased IL-2 expression. Mechanistically, we showed that Prox1 inhibited the IL-2 promoter activity, and associated with the minimal IL-2 promoter. Prox1 repressed the nuclear factor of activated T cells 2 (NFAT2)-dependent transactivation of IL-2 gene by physically binding to NFAT2. The N-terminal region of Prox1 was essential for the binding and repression. In summary, our findings established Prox1 as a negative regulator in IL-2 gene expression through the direct interaction with NFAT2.

Dynamic Expression of Sox2, Gata3, and Prox1 during Primary Auditory Neuron Development in the Mammalian Cochlea.

Primary auditory neurons (PANs) connect cochlear sensory hair cells in the mammalian inner ear to cochlear nucleus neurons in the brainstem. PANs develop from neuroblasts delaminated from the proneurosensory domain of the otocyst and keep maturing until the onset of hearing after birth. There are two types of PANs: type I, which innervate the inner hair cells (IHCs), and type II, which innervate the outer hair cells (OHCs). Glial cells surrounding these neurons originate from neural crest cells and migrate to the spiral ganglion. Several transcription factors are known to regulate the development and differentiation of PANs. Here we systematically examined the spatiotemporal expression of five transcription factors: Sox2, Sox10, Gata3, Mafb, and Prox1 from early delamination at embryonic day (E) 10.5 to adult. We found that Sox2 and Sox10 were initially expressed in the proneurosensory cells in the otocyst (E10.5). By E12.75 both Sox2 and Sox10 were downregulated in the developing PANs; however, Sox2 expression transiently increased in the neurons around birth. Furthermore, both Sox2 and Sox10 continued to be expressed in spiral ganglion glial cells. We also show that Gata3 and Prox1 were first expressed in all developing neurons, followed by a decrease in expression of Gata3 and Mafb in type I PANs and Prox1 in type II PANs as they matured. Moreover, we describe two subtypes of type II neurons based on Peripherin expression. These results suggest that Sox2, Gata3 and Prox1 play a role during neurogenesis as well as maturation of the PANs.

Overexpression of Prox1 Induces the Differentiation of Human Adipose-Derived Stem Cells into Lymphatic Endothelial-Like Cells In Vitro.

Constant levels of homeobox transcription factor Prox1 expression are required throughout the life of lymphatic endothelial cells (LECs) to maintain their differentiated identity. Recent studies have demonstrated that using human LECs for cell transplantation therapy may improve secondary lymphedema in a nude rat model. However, the application is currently limited by the low yield of LECs. In this study, Prox1 was overexpressed in human adipose tissue-derived stem cells (hADSCs) by using the transfection of lentiviral vectors to induce the differentiation of hADSCs to LECs. After 14 days of Prox1 overexpression, flow cytometry analysis found that the expression of LEC-specific markers such as Podoplanin and VEGFR3, along with the endothelial cell (EC) marker CD31, on Prox1-overexpressed hADSCs was significantly increased; however, the expression of mesenchymal stem cell markers, such as CD29, CD44, and CD90, was substantially reduced. In addition, the mRNA levels of the LEC-specific markers, such as Prox1, Podoplanin, LYVE1, and VEGFR3, in Prox1-overexpressed hADSCs were significantly increased at day 7 and maintained a continuously increased expression level for 28 observation days, according to real-time reverse transcriptase-polymerase chain reaction results. Western blotting and immunofluorescence staining results further confirmed that overexpression of Prox1 in hADSCs significantly increased the protein levels of Podoplanin, LYVE1, and VEGFR3, as well as those of the EC markers such as VWF and CD144, at day 14. Moreover, these differentiated cells were found to form tube-like structures in matrigel, measured by the tube formation assay. These findings suggested that overexpression of Prox1 in hADSCs successfully induced the differentiation of hADSCs into stable lymphatic endothelial-like cells. This study achieved a long-lasting expression of Prox1 in lymphatic endothelial-like cells, and it provided a potentially useful approach for developing novel therapies for limb lymphedema and lymphatic system-related diseases.