Generation of Leydig-like cells: approaches, characterization, and challenges.

Testosterone production by Leydig cells (LCs) plays a crucial role in male reproduction. The functional degeneration of LCs can cause testosterone deficiency, ultimately resulting in primary male hypogonadism. Transplantation of exogenous LCs with the ability to produce testosterone in response to the regulation of the hypothalamus-pituitary-gonad axis could be a promising alternative option to treat male primary hypogonadism. Recent studies have shown that it is possible to generate Leydig-like cells from stem cells by various approaches. In addition, somatic cells, such as embryonic or adult fibroblasts, have also been successfully reprogrammed into Leydig-like cells. In this review, we summarized the recent advances in the generation of Leydig-like cells, with an emphasis on comparing the effectiveness and safety of different protocols used and the cells generated. By further analyzing the characteristics of Leydig-like cells generated from fibroblasts based on small signaling molecules and regulatory factors, we found that although the cells may produce testosterone, they are significantly different from real LCs. For future in vivo applications, it is important that the steroidogenic cells generated be evaluated not only for their steroidogenic functions but also for their overall cell metabolic state by proteomics or transcriptomic tools.

Human Basic Fibroblast Growth Factor Inhibits Tau Phosphorylation via the PI3K/Akt-GSK3ß Signaling Pathway in a 6-Hydroxydopamine-Induced Model of Parkinson's Disease.

BACKGROUND: Basic fibroblast growth factor (bFGF) has been increasingly investigated due to its neuroprotection in neurodegenerative disorders. Because there are still no cures for any of these disorders, it is crucial to identify new therapeutic targets and screen potential drugs. The increased phosphorylation of tau at Ser396 leads to intracellular tau accumulation, which forms neurofibrillary tangles in Parkinson's disease (PD). In this study, neuroprotection by bFGF was observed, and the mechanisms related to its regulation of phosphorylated tau were investigated. METHODS: bFGF-loaded liposome carriers were intranasally administered to rats. The neuroprotective effects of bFGF were assessed in a PD model induced by 6-hydroxydopamine (6-OHDA) in vivo and in vitro. The phosphorylation of tau was measured, and the PI3K/Akt-GSK3ß signaling pathway was investigated. RESULTS: Our study demonstrated that liposomes markedly assisted in the delivery of bFGF to the striatum and substantia nigra of rats and enhanced the neuroprotective effects of bFGF on dopaminergic neurons. bFGF treatment significantly ameliorated the behavioral deficits induced by 6-OHDA, rescued the loss of tyrosine hydroxylase-positive neurons and increased the number of Nissl bodies. bFGF reduced the phosphorylation of tau and GSK3ß and increased the phosphorylation of PI3K/Akt. CONCLUSION: Liposomes markedly assisted in the delivery of bFGF to the brain and enhanced the neuroprotective effects of bFGF by inhibiting the phosphorylation of tau. bFGF down-regulated the phosphorylation of tau by increasing the phosphorylation of GSK3ß via the PI3K/Akt signaling pathway. These findings provide a new vision of bFGF as a potential therapy for PD.

Expression of curcin-transferrin receptor binding peptide fusion protein and its anti-tumor activity.

Curcin can inhibit the proliferation of tumor cells and promote tumor cell apoptosis, but the cytotoxicity of curcin is not selective for tumors or normal cells. In order to enhance the targeting of the anti-tumor ability of curcin, a transferrin receptor (TfR) binding peptide, TfRBP9, was fused with curcin. The curcin-TfRBP9 gene was cloned into pQE-30 and the recombinant vector pQE-30-curcin-TfRBP9 was established. Then the recombinant vector pQE-30-curcin-TfRBP9 was transferred into Escherichia coli M15. After being induced by 0.5mM IPTG for 6h at 37°C, the expressed quantity of the recombinant protein was about 30% of the total protein. Recombinant curcin-TfRBP9 was expressed in the form of an inclusion body. After dissolution, purification and renaturation, the purity of the recombinant curcin-TfRBP9 reached 95%. Immunofluorescence analysis showed that the TfRBP9 significantly enhanced the ability of the curcin binding to HepG2, and was enriched in the cytoplasm. The curcin-TfRBP9 fusion protein had significant proliferation inhibition effects on the HepG2 cells that over-expressed transferrin receptors, had lower inhibitory effects on the SKBR-3 cells that expressed low transferrin receptors, and had the lowest inhibitory effects on the LO-2 cells that were normal human liver cells. Compared with curcin, the curcin-TfRBP9 induced higher apoptosis rates in the HepG2 cells.

The effect of transforming growth factor-beta1 on nasopharyngeal carcinoma cells: insensitive to cell growth but functional to TGF-beta/Smad pathway.

OBJECTIVES: This study explored the response of nasopharyngeal carcinoma cells to TGF-beta1-induced growth suppression and investigated the roles of the TGF-beta/Smad signaling pathway in nasopharyngeal carcinoma cells. METHODS: The cells of nasopharyngeal carcinoma cell line CNE2 were treated with TGF-beta1. The growth responses of CNE2 cells were analyzed by MTT assay. The mRNA expression and protein subcellular localization of the TGF-beta/Smad signaling components in the CNE2 were determined by real time RT-PCR and immunocytochemical analysis. RESULTS: We found that the growth of CNE2 cells was not suppressed by TGF-beta1. The signaling proteins TbetaRII, Smad 7 were expressed normally, while Smad2, Smad3, and Smad4 increased significantly at the mRNA level. TGF-beta type II receptor and Smad7 had no change compared to the normal nasopharyngeal epithelial cells. In addition, Smad2 was phosphorylated to pSmad2, and the activated pSmad2 translocated into the nucleus from the cytoplasm, while the inhibitory Smad-Smad7 translocated from the nucleus to the cytoplasm after TGF-beta1 stimulation. CONCLUSION: The results suggested that CNE2 cells are not sensitive to growth suppression by TGF-beta1, but the TGF-beta/Smad signaling transduction is functional. Further work is needed to address a more detailed spectrum of the TGF-beta/Smad signaling pathway in CNE2 cells.

Retina protective effect of acidic fibroblast growth factor after canceling its mitogenic activity.

PURPOSE: The aim of this study was to investigate the effect of mutant of acidic fibroblast growth factor (MaFGF) on N-methyl-N-nitrosourea (MNU)-induced retinal degeneration in Sprague-Dawley rats. METHODS: Fifty (50)-day-old female Sprague-Dawley rats were given a single intraperitoneal injection of normal saline (NS) or 60 mg x kg(-1) body weight of MNU, and then NS or different doses of MaFGF were injected intravitreally twice at 0 and 12 h after NS or MNU treatment. After NS or MNU treatment for different times, the apoptotic index of the photoreceptor cell was detected by TUNEL labeling, whereas the mRNA expressions and the protein levels of antiapoptotic Bcl-2 and proapoptotic Bax were determined by reverse transcriptase polymerase chain reaction and Western blotting, respectively. Retinal damage was evaluated based on retinal thickness. RESULTS: MNU-induced retinal damage was partially protected by MaFGF in a dose-independent manner in rats. MaFGF at doses of 1.25 and 2.5 microg could partially suppress photoreceptor cell loss, whereas MaFGF at a dose of 5.0 mug had no protective effect on photoreceptor cell. The apoptotic index at 24 h post-MNU in the peripheral retina was 38.1 +/- 3.6%, whereas 1.25 and 2.5 mug MaFGF markedly reduced it to 27.5 +/- 2.0 and 21.1 +/- 1.9% (P = <0.001), respectively. As compared with the MNU-treated group, MaFGF significantly upregulated the expression of Bcl-2 mRNA and protein and downregulated the expression of Bax mRNA and protein (P = <0.001). CONCLUSION: MaFGF could counteract MNU-induced retinal damage and may be a therapeutic agent for the treatment of retinal degeneration.

[Effect and mechanism of action of non-mitogenetic human acidic fibroblast growth factor on the mitogenic activity of the mammary tumor cell].

AIM: To compare the effects of the non-mitogenetic human acidic fibroblast growth factor (nmhaFGF) and the human acidic fibroblast growth factor (haFGF) on the proliferation and MAPK signal transduction pathway of the malignant tumor cell and to study the clinical safety of nmhaFGF. METHODS: The mammary tumor cells (MCF-7) were treated with haFGF and nmhaFGF separately. The mitogenic activities of both haFGF and nmhaFGF were detected by MTT method and the cell cycle was analyzed by flow cytometer (FCM). The expression levels of the signal proteins, Grb2 (growth factor receptor bound 2) and ERK1/2 (extracellular signal-regulated kinase 1/2), were detected by semi-quantitative Western blotting method. RESULTS: The mitogenic activity of nmhaFGF was obviously lower than that of haFGF. The activity of nmhaFGF was weaker than that of the haFGF. The ratio of G1/G0, G2/M of haFGF was markedly lower than that of nmhaFGF and control group, and was reverse in S phase. The expression levels of both Grb2 and ERK1/2 of the nmhaFGF treated group were lower than that of the haFGF treated group and approaching the control group. CONCLUSION: The mitogenic activity of the nmhaFGF decreased remarkably. Its mechanism probably via down-regulation of the expression of the signal moleculars, MAPK-ERK1/2 and Grb2.

A vascular cell-restricted RhoGAP, p73RhoGAP, is a key regulator of angiogenesis.

Angiogenesis is a major therapeutic target. Ideal drug targets are genes expressed only in endothelial cells (ECs) or only during the angiogenic process. Here, we describe a gene, p73RhoGAP (p73), that has both of these properties. By using a PCR-based subtraction-hybridization approach to clone cDNAs from ECs undergoing capillary-tube formation, we identified a RhoGAP member, p73. p73 displays GTPase activity to Rho but not to Rac or Cdc42. Knockdown of p73 protein, achieved by adenovirus delivery of p73 antisense and by small interfering RNA into ECs, demonstrated the importance of this protein in EC function. Under such conditions, EC migration, proliferation, and capillary-tube formation were inhibited. Furthermore, angiogenesis in vivo was also inhibited by antisense p73. A mutant R82A alteration achieved a similar phenotype in vitro to the antisense, demonstrating the importance of the GTPase-activating protein activity to p73 function. Expression profiling of p73 shows that it is vascular cell-selective, being highly expressed in ECs and smooth-muscle cells but not in other cell types. Finally, we show that the mRNA of p73 is up-regulated in an angiogenic milieu with little or no regulation seen under nonangiogenic conditions. p73, a vascular cell-specific GTPase-activating protein, is an important modulator of angiogenesis and displays many of features that make it worthy of being a drug target.