Opioid and Cannabinoid Receptors Share a Common Pool of GTP-Binding Proteins in Cotransfected Cells, But Not in Cells Which Endogenously Coexpress the Receptors

1. Opioid (, , ) and cannabinoid (CB1, CB2) receptors are coupled mainly toG_i/G_o GTP-binding proteins. The goal of the present study was to determine whether different subtypes of opioid and cannabinoid receptors, when coexpressed in the same cell, share a common reservoir, or utilize different pools, of G proteins.2. The stimulation of [³⁵S]GTPS binding by selective opioid and cannabinoid agonists was tested in transiently transfected COS-7 cells, as well as in neuroblastoma cell lines. In COS-7 cells, cotransfection of - and -opioid receptors led to stimulation of [³⁵S]GTPS binding by either -selective (DAMGO) or -selective (DPDPE) agonists. The combined effect of the two agonists was similar to the effect of either DAMGO or DPDPE alone, suggesting the activation of a common G-protein reservoir by the two receptor subtypes.3. The same phenomenon was observed when COS-7 cells were cotransfected with CB1 cannabinoid receptors and either - or -opioid receptors.4. On the other hand, in N18TG2 neuroblastoma cells, which endogenously coexpress CB1 and -opioid receptors, as well as in SK-N-SH neuroblastoma cells, which coexpress - and -opioid receptors, the combined effects of the various agonists (the selective cannabinoid DALN and the selective opioids DPDPE and DAMGO) were additive, implying the activation of different pools of G proteins by each receptorsubtype.5. These results suggest a fundamental difference between native and artificially transfected cells regarding the compartmentalization of receptors and GTP-binding proteins.     

MiR-124 Promote Neurogenic Transdifferentiation of Adipose Derived Mesenchymal Stromal Cells Partly through RhoA/ROCK1, but Not ROCK2 Signaling Pathway

Objective

Some recent studies suggest that multiple miRNAs might regulate neurogenic transdifferentiation of mesenchymal stromal cells (MSCs). In the present study, we hypothesized that the miR-124 can repress the expression of RhoA upon the neurogenesis of adipose derived MSCs (ADMSCs).

Methods

MiRNA expression dynamics during neurogenic transdifferentiation of ADMSCs were measured. The expression of neuron-specific enolase (NSE), Tuj-1 (Neuron-specific class III beta-tubulin) and glial fibrillary acidic protein (GFAP), as well as electrophysiological properties, were detected after neurogenic transdifferentiation. The targeting of miR-124 over RhoA was verified by dual luciferase assay, qRT-PCR and western blot. The functions of miR-124 and the RhoA/ROCK signaling pathway were studied using gain and loss of function experiments in vitro.

Results

MiR-124 is significantly upregulated during neurogenic transdifferentiation of ADMSCs. Knockdown of endogenous miR-124 hampered neurogenic transdifferentiation and the acquired electrophysiological properties. MiR-124 could directly target RHOA mRNA and repress its expression, through which it increased the proportion of transdifferentiated (transdiff.) cells with positive NSE, Tuj-1 and GFAP. RhoA/ROCK1, but not ROCK2 is a downstream signaling pathway of miR-124 in the process of transdifferentiation.

Conclusion

MiR-124 is an important miRNA modulating neurogenic transdifferentiation of ADMSCs at least partly via the miR-124/RhoA/ROCK1 signaling pathway. These findings provided some fundamental information for future use of ADMSCs as an agent for regenerative medicine and cell therapy for neurological diseases.     

Effects of BMP-2 and FGF2 on the Osteogenesis of Bone Marrow-Derived Mesenchymal Stem Cells in Hindlimb-Unloaded Rats

Hindlimb unloading, as a simulation of microgravity, decreases the osteogenic potential of mesenchymal stem cells (MSCs) from hindlimb femur of rat. We simulated the microgravity by 28-day of hindlimb unloading for male Sprague–Dawley rat, and performed intramuscular injection of BMP-2 and FGF2 at a given interval during hindlimb unloading. Then, the bone marrow (BM) was collected from hindlimb femur of rat. MSCs were isolated from BM, cultured for four passages, and then induced for osteogenesis. The results revealed that the hindlimb unloading decreased the osteogenic potential of MSCs and also the expression of osteoblast gene marker mRNAs in cells induced by osteogenic conditions. Hindlimb unloading for 28 days resulted in the decrease of vinculin-containing focal adhesion in MSCs. During hindlimb unloading, the interval intramuscular injection of BMP-2 or FGF2 alone could increase the osteogenic potential of MSCs and the expression of osteoblast gene marker mRNA. However, the effect of BMP-2 or FGF2 injection alone was significantly lower than that of combination injection of both factors. The further examination showed that the intramuscular injection of BMP-2 promoted the expression of Runx2 mRNA and that the intramuscular injection of FGF2 increased the phosphorylation of ERK and Runx2. Nevertheless, the intramuscular injection of any factor could not increase the formation of vinculin-containing focal adhesions in MSCs. This suggests that BMP-2 should increase the expression of Runx2, and that the activation of Runx2 should be promoted by the FGF2 signaling pathway which activated ERK/Runx2. The activation of this signaling pathway should not lie on the formation of vinculin-containing focal adhesions.     

Identification of a novel FGF, FGF-21, preferentially expressed in the liver

We isolated cDNA encoding a novel FGF (210 amino acids) from mouse embryos. As this is the 21st documented FGF, we tentatively term it FGF-21. FGF-21 has a hydrophobic amino terminus ( approximately 30 amino acids), which is a typical signal sequence, and appears to be a secreted protein. The expression of FGF-21 mRNA in mouse adult tissues was examined by Northern blotting analysis. FGF-21 mRNA was most abundantly expressed in the liver, and also expressed in the thymus at lower levels. We also isolated human cDNA encoding FGF-21 (209 amino acids). Human FGF-21 is highly identical ( approximately 75% amino acid identity) to mouse FGF-21. Among human FGF family members, FGF-21 is most similar ( approximately 35% amino acid identity) to FGF-19.     

Structural basis for interaction of FGF-1, FGF-2, and FGF-7 with different heparan sulfate motifs.

Stromal cell-derived FGF-7 binds and activates only the resident FGFR2IIIb in epithelial cells while FGF-1 and FGF-2 exhibit a broader interaction with multiple isoforms of FGFR. Here we report the structure of FGF-7 that has been solved to 3.1 A resolution by molecular replacement with the structure of a dual function chimera of FGF-7 and FGF-1 (FGF-7/1) which was resolved to 2.3 A. Comparison of the FGF-7 structure to that of FGF-1 and FGF-2 revealed the strongly conserved Calpha backbone among the three FGF polypeptides and the surface hydrophobic patch that forms the primary receptor-binding domain. In contrast, a decrease and dispersion of the positive surface charge density characterized the heparin-binding domain of FGF-7 defined by homology to that of FGF-1 and FGF-2 in complexes with heparin. A simple heparin hexasaccharide that cocrystallized with FGF-1 and FGF-2 and protected both against protease in solution failed to exhibit the same properties with FGF-7. In contrast to FGF-1 and FGF-2, protection of FGF-7 was enhanced by heparin oligosaccharides of increased length with those exhibiting a 3-O-sulfate being the most effective. Protection of FGF-7 required interaction with specifically the fraction of crude heparin retained on antithrombin affinity columns. Conversely, heparin enriched by affinity for immobilized FGF-7 exhibited anti-factor Xa activity similar to that purified on an antithrombin affinity matrix. In contrast, an FGF-1 affinity matrix enriched the fraction of crude heparin with low anti-factor Xa activity. The results provide a structural basis to suggest that the unique FGF-7 heparin-binding (HB) domain underlies a specific restriction in respect to composition and length of the heparan sulfate motif that may impact specificity of localization, stability, and trafficking of FGF-7 in the microenvironment, and formation and activation of the FGFR2IIIb kinase signaling complex in epithelial cells.     

Regulation of osteoblast, chondrocyte, and osteoclast functions by fibroblast growth factor (FGF)-18 in comparison with FGF-2 and FGF-10.

This study investigated the actions of fibroblast growth factor (FGF)-18, a novel member of the FGF family, on osteoblasts, chondrocytes, and osteoclasts and compared them with those of FGF-2 and FGF-10. FGF-18 stimulated the proliferation of cultured mouse primary osteoblasts, osteoblastic MC3T3-E1 cells, primary chondrocytes, and prechondrocytic ATDC5 cells, although it inhibited the differentiation and matrix synthesis of these cells. FGF-18 up-regulated the phosphorylation of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and up-regulated the phosphorylation of p38 mitogen-activated protein kinase only in chondrocytes. FGF-18 mitogenic actions were blocked by a specific inhibitor of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and by a specific inhibitor of p38 mitogen-activated protein kinase in chondrocytes. With regard to the action of FGF-18 on bone resorption, FGF-18 not only induced osteoclast formation through receptor activator of nuclear factor-kappaB ligand and cyclooxygenase-2 but also stimulated osteoclast function to form resorbed pits on a dentine slice in the mouse coculture system. All these effects of FGF-18 bore a close resemblance to those of FGF-2, whereas FGF-10 affects none of these cells. FGF-18 may therefore compensate for the action of FGF-2 on bone and cartilage.     

成纤维生长因子-2与肝细胞生长因子诱导骨髓间充质干细胞分化为肝细胞样细胞的作用比较

比较成纤维生长因子-2(FGF-2)与肝细胞生长因子(HGF)诱导大鼠骨髓间充质干细胞(BM-MSCs)向肝细胞分化的能力,并进一步的研究BM-MSCs诱导成肝细胞所需的最佳诱导因子以及其用量。体外获取、培养大鼠BM-MSCs,将第3代BM-MSCs采用不同剂量的FGF-2和HGF诱导。诱导后,在显微镜下观察细胞形态学的改变;Shiff染色法检测糖原的分泌;ELISA法检测AFP的分泌;免疫细胞化学法检测白蛋白和CK19的分泌。诱导后BM-MSCs由梭形向多角形、卵圆形方向变化,AFP第3天就有分泌,第12天达到高峰,以后逐渐减少;白蛋白、CK19和糖原第12天即有阳性表达,以后随着诱导时间的延长阳性率逐渐升高。第2组和第4组比其他组分泌的白蛋白、CK19和糖原均多。FGF-2比HGF具有更强的诱导BM-MSCs向肝细胞分化的能力。     

FGF-2对人骨髓间充质干细胞增殖和向成骨细胞分化的影响

探讨体外培养条件下,成纤维细胞生长因子-2（FGF-2）和地塞米松（Dex）对第7代人骨髓间充质干细胞（MSCs）增殖和向成骨细胞分化的作用以及两者联合使用的效应。MSCs经含FGF-2或／和Dex的培养液作用后,于不同时间采用MTT法测定细胞增殖情况;对硝基苯磷酸（pNPP）法测定碱性磷酸酶（ALP）活性;ELISA法测定骨钙蛋白（OC）含量;茜素红S染色法对沉积的钙盐进行染色。发现：（1）FGF-2组细胞的生长速度为对照组的1．31倍,Dex／FGF-2组细胞的生长速度为FGF-2组的1．12倍。（2）Dex组的ALP活性、OC含量和细胞外基质钙盐沉积分别为对照组的17．0倍、2．12倍和10．56倍,并能形成成熟的羟基磷灰石（HA）结晶和骨结节;FGF-2组的ALP活性比对照组降低了76．7％,虽然OC含量、钙盐沉积增加,但不能形成成熟的HA结晶和骨结节;FGF-2对Dex诱导的ALP活性增加和HA结晶形成有拮抗作用。由此证明：（1）FGF-2可促进MSCs的增殖,Dex对MSCs的增殖无明显作用;Dex能增强FGF-2对MSCs的促增殖效应。（2）Dex可使MSCs分化为成熟的成骨细胞,是一个有效的成骨细胞分化诱导剂;FGF-2可使MSCs分化为未成熟的成骨细胞;FGF-2拮抗Dex诱导MSCs分化为成熟的成骨细胞。     

High Expression of HuR in Cytoplasm,but Not Nuclei,Is Associated with Malignant Aggressiveness and Prognosis in Bladder Cancer

Introduction

Human antigen R (HuR) regulates the stability of mRNA and is associated with cell proliferation, angiogenesis, and lymphangiogenesis. However, the clinical significance and pathological role of HuR in bladder cancer remains unclear. The main objective of this investigation was to clarify the relationships between HuR expression and clinical significance and cancer cell proliferation, angiogenesis, lymphangiogenesis, and expressions of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF)-A, -C, and -D.

Methods

All expressions were examined by immunohistochemical techniques in 122 formalin-fixed specimens of bladder cancer patients. HuR expression was evaluated separately with cytoplasmic and nuclear staining. Cell proliferation, angiogenesis and lymphangiogenesis were measured as the percentage of Ki-67-positive cell (proliferation index, PI), CD34-stained vessels (microvessel density, MVD), and D2-40-stained vessels (lymph vessel density, LVD). Relationships between each HuR expression and clinicopathological features, prognosis, and expressions of COX-2 and VEGFs were analyzed by multi-variate analyses. HuR expression was also investigated in 10 mice of N-Butyl-N-[4-hydroxybutil] nitrosamine (BBN) induced bladder cancer model.

Results

In human tissues, high cytoplasmic expression was seen in 5% and 25.4% of normal and cancer cells, respectively. Nuclear HuR expression bore no significant relationship to any pathological features. However, cytoplasmic HuR expression appeared positively associated with pT stage and grade (P<0.001). In mouse tissues, similar trends were confirmed. Cytoplasmic expression correlated with PI, MVD, and LVD, as well as expression of VEGF-A and -C, but not VEGF-D. High cytoplasmic expression of HuR was a significant predictor of metastasis and cause-specific survival, and was identified as a prognostic correlative factor for metastasis (hazard ratio, 4.75; P = 0.028) in a multivariate analysis model that included pathological features.

Conclusions

Cytoplasmic HuR appears to play important roles in cell proliferation, progression, and survival of bladder cancer patients. Its expression was associated with angiogenesis, lymphangiogenesis, and expressions of VEGF-A and –C.     

Cancer-Associated Fibroblasts in a Human HEp-2 Established Laryngeal Xenografted Tumor Are Not Derived from Cancer Cells through Epithelial-Mesenchymal Transition,Phenotypically Activated but Karyotypically Normal

Cancer-associated fibroblasts (CAFs) play a crucial role in cancer progression and even initiation. However, the origins of CAFs in various cancer types remain controversial, and one of the important hypothesized origins is through epithelial-mesenchymal transition (EMT) from cancer cells. In this study, we investigated whether the HEp-2 laryngeal cancer cells are able to generate CAFs via EMT during tumor formation, which is now still unknown. The laryngeal xenografted tumor model was established by inoculating the HEp-2 laryngeal cancer cell line in nude mice. Primary cultured CAFs from the tumor nodules and matched normal fibroblasts (NFs) from the adjacent connective tissues were subcultured, purified, and verified by immunofluorescence. Migration, invasion, and proliferation potentials were compared between the CAFs and NFs. A co-culture of CAFs with HEp-2 cells and a co-injection of CAFs with HEp-2 cells in nude mice were performed to examine the cancer-promoting potential of CAFs to further verify their identity. Karyotypic analyses of the CAFs, NFs, and HEp-2 cells were conducted. A co-culture of NFs with HEp-2 cells was also performed to examine the expression of activated markers of CAFs. A pathological examination confirmed that the laryngeal xenografted tumor model was successfully established, containing abundant CAFs. Immunocytochemical staining verified the purities and identities of the CAFs and NFs. Although the CAFs manifested higher migration, invasion, proliferation, and cancer-promoting capacities compared with the NFs, an analysis of chromosomes revealed that both the CAFs and NFs showed typical normal mouse karyotypes. In addition, the NFs co-cultured with HEp-2 cells did not show induced expressions of activated markers of CAFs. Our findings reveal that the CAFs in the HEp-2 established laryngeal xenografted tumor are not of laryngeal cancer origin but of mouse origin, indicating that the HEp-2 laryngeal cancer cells cannot generate their own CAFs via EMT in this model.     

APT1, but Not APT2, Codes for a Functional Adenine Phosphoribosyltransferase in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has two separate genes (APT1 and APT2) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT). However, genetic analysis indicated that only APT1 could code for a complementing activity. Cloning and expression of both the APT1 and APT2 genes in Escherichia coli showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity. Northern and Western blot analyses demonstrated that only APT1 was transcribed and translated under normal physiological conditions in yeast. Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event. We conclude that APT1 is the functional gene in S. cerevisiae and that APT2 is a pseudogene.     

Loss of Syndecan-1 Induces a Pro-inflammatory Phenotype in Endothelial Cells with a Dysregulated Response to Atheroprotective Flow

Fluid shear stresses are potent regulators of vascular homeostasis and powerful determinants of vascular disease progression. The glycocalyx is a layer of glycoaminoglycans, proteoglycans, and glycoproteins that lines the luminal surface of arteries. The glycocalyx interacts directly with hemodynamic forces from blood flow and, consequently, is a prime candidate for the mechanosensing of fluidic shear stresses. Here, we investigated the role of the glycocalyx component syndecan-1 (sdc-1) in controlling the shear stress-induced signaling and flow-mediated phenotypic modulation in endothelial cells. We found that knock-out of sdc-1 abolished several key early signaling events of endothelial cells in response to shear stress including the phosphorylation of Akt, the formation of a spatial gradient in paxillin phosphorylation, and the activation of RhoA. After exposure to atheroprotective flow, we found that sdc-1 knock-out endothelial cells had a phenotypic shift to an inflammatory/pro-atherosclerotic phenotype in contrast to the atheroprotective phenotype of wild type cells. Consistent with these findings, we found increased leukocyte adhesion to sdc-1 knock-out endothelial cells in vitro that was reduced by re-expression of sdc-1. In vivo, we found increased leukocyte recruitment and vascular permeability/inflammation in sdc-1 knock-out mice. Taken together, our studies support a key role for sdc-1 in endothelial mechanosensing and regulation of endothelial phenotype.