Ultrasound-induced activation of Wnt signaling in human MG-63 osteoblastic cells

The benefit from an ultrasound (US) exposure for fracture healing has been clearly shown. However, the molecular mechanisms behind this effect are not fully known. Recently, the canonical Wnt signaling pathway has been recognized as one of the essential regulators of osteoblastogenesis and bone mass, and thereby considered crucial for bone health. Mechanical loading and fluid shear stress have been reported to activate the canonical Wnt signaling pathway in bone cells, but previous reports on the effects of therapeutic US on Wnt signaling in general or in bone, in particular, have not been published yet. Therefore, activation of Wnt signaling pathway was assayed in human osteoblastic cells, and indeed, this pathway was found to be activated in MG-63 cells through the phosphoinositol 3-kinase/Akt (PI3K/Akt) and mTOR cascades following a single 10 min US exposure (2 W, 1.035 MHz). In addition to the reporter assay results, the Wnt pathway activation was also observed as nuclear localization of β-catenin. Wnt activation showed also temperature dependence at elevated temperatures, and the expression of canonical Wnt ligands was induced under the thermal exposures. However, existence of a specific, non-thermal US component was evident as well, perhaps evidence of a potential dual action of therapeutic US on bone. Neither US nor heat exposures affected cell viability in our experiments. In summary, this is the first study to report that Wnt signaling cascade, important for osteoblast function and bone health, is one of the pathways activated by therapeutic US as well as by hyperthermia in human osteoblastic cells. Our results provide evidence for the potential molecular mechanisms behind the beneficial effects of US on fracture healing. Combinations of US, heat, and possible pharmacological treatment could provide useful flexibility for clinical cases in treating various bone disorders.     

Prostaglandin-dependent activation of ERK mediates cell proliferation induced by transforming growth factor beta in mouse osteoblastic cells

Transforming growth factor beta (TGF(beta)) is a major coupling factor for bone turnover and is known to stimulate osteoblastic proliferation. Recent information indicates that, in addition to the Smad pathway, TGF(beta) also activates MAP kinases in osteoblastic cells. The role of these signaling cascades in cell proliferation induced by TGF(beta) as well as the cellular and molecular mechanisms of their activation by TGF(beta) has been investigated in this study. In MC3T3-E1 cells, TGF(beta) enhanced cell proliferation by about 2-fold and induced activation of the three MAP kinases, extracellular regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Surprisingly, however, whereas activation of Smad2 was rapid and maximal after 15-min incubation, activation of MAP kinases was delayed with p38 stimulation detected after 1-h exposure and activation of ERK and JNK after 3 h, suggesting indirect activation of MAP kinases by TGF(beta). Among factors known to be released in response to TGF(beta) in osteoblastic cells and influence their growth, prostaglandins (PGs) were good candidates that were further investigated for mediating TGF(beta)-induced activation of MAP kinases and cell proliferation. Indomethacin, a selective inhibitor of PG synthesis, completely blunted cell proliferation induced by TGF(beta) and markedly reduced activation of MAP kinases without influencing Smad2 phosphorylation. EP4A, a specific PGE2 receptor antagonist, also blunted TGF(beta)-induced osteoblastic proliferation. In addition to these effects, PGE2 rapidly activated MAP kinases in MC3T3-E1 cells and increased cell proliferation by about 2-fold. The role of each MAP kinases in mediating TGF(beta)- and PGE2-induced cell proliferation was investigated using selective inhibitors. U0126, a specific inhibitor of the ERK pathway, completely blocked both TGF(beta)- and PGE2-induced cell proliferation whereas SB203580 and SP600125, which are selective inhibitors of, respectively, p38 and JNK pathways, had no effect. Finally, the effect of PGE2 on activation of ERK was mimicked by phorbol esters and not by forskolin, and was associated with activation of protein kinase C. This latter effect and the stimulation of ERK induced by PGE2 were completely blocked by a specific inhibitor of PKC. In conclusion, data presented in this study strongly suggest that the local release of PGE2 is involved in cell proliferation induced by TGF(beta) in osteoblastic cells. This effect is mediated by the ERK pathway activated by a PKC-dependent mechanism.     

成人成骨细胞和MG-63细胞株孕激素受体的表达

目的 观察成人成骨细胞和MG-63细胞株孕激素受体(progesterone receptor，PR)亚型表达的情况。方法 用改良胶原酶消化法从正常成人松质骨中分离成骨细胞，观察细胞形态，钙钴法行碱性磷酸酶染色，Van Gieson法行Ⅰ型胶原染色，茜素红行矿化结节染色；半定量RT-PCR检测骨钙素和PR亚型mRNA表达；Western blot测定PR蛋白质表达。结果 所分离培养的细胞具有成骨细胞的形态特征，保持了其在体内的功能；人成骨细胞和MG-63细胞株均表达PRA、PRB mRNA和蛋白质。结论 人成骨细胞和MG-63细胞株可能受孕激素的影响。     

bFGF 、TGFβ-1在膀胱出口梗阻患者逼尿肌细胞中的表达

目的 观察膀胱出口梗阻（BOO）后膀胱平滑肌细胞碱性成纤维细胞生长因子（bFGF）、转化生长因子（TGFβ-1）表达的变化,探讨生长因子在BOO后膀胱平滑肌细胞继发改变中的作用。方法 应用逆转录－聚合酶链反应（RT－PCR）和免疫组织化学SP法在mRNA和蛋白水平检测bFGF、TGFβ-1在30例BOO及4例非BOO膀胱平滑肌组织中的表达。结果 BOO组与非BOO组膀胱平滑肌组织均有bFGF mRNA表达,BO组表达水平高于非BOO组,P＜0．01;TGFβ-1 mRNA在两组未见表达。结论 BOO 后磅胱平滑肌的继发改变与bFGF mRNA的表达有关。     

Interaction of epidermal growth factor and transforming growth factor beta in human prostatic epithelial cells in culture.

The present study was conducted to study the interaction between epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) in benign human prostatic epithelial cells in culture. Primary cultures of human prostatic epithelial cells were grown in complete WAJC, which consisted of WAJC-404 medium and, in addition to other defined additives, EGF and bovine pituitary extract (BPE). Incomplete WAJC contained the same composition except EGF and BPE were deleted. TGF-beta was added into media at concentrations of 0, 0.1, and 1.0 ng/ml. When cells were grown in complete WAJC, they proliferated rapidly. Cell proliferation was greatly suppressed when incomplete WAJC was used. Addition of TGF-beta to these cultures caused a significant reduction in the final cell number when either complete WAJC or incomplete WAJC was used. In additional experiments, cells were prelabeled with 3H-thymidine for 72 hr prior to treatment with TGF-beta. The percentage of radioactivity released into the medium at the end of a 6-day culture was used as an indication of the extent of cell death. Trypan blue exclusion test was also used to assess the extent of cell death. Addition of TGF-beta into complete WAJC did not significantly affect the extent of cell death beyond what was considered as the result of normal cellular turnover. Addition of TGF-beta into incomplete WAJC, however, caused a significant increase in the percent of cell death in the culture. These results demonstrated an interaction between EGF and TGF-beta in proliferation and cell death in human prostatic epithelia in culture. In the presence of EGF alone in the culture medium, prostatic epithelial cells were stimulated to proliferate. The rate of proliferation was greatly diminished when EGF was deleted from the medium or when TGF-beta was added in the presence of EGF. Finally, cell death was induced when TGF-beta was added into the medium in the absence of EGF.     

Effect of transforming growth factor beta and basic fibroblast growth factor on steroid-impaired healing intestinal wounds.

A longitudinal intestinal wound model in the pig was used to assess the effect of parenteral steroids (betamethasone 12 mg 50 kg-1 intramuscularly twice daily) on breaking load. Steroid treatment significantly decreased the breaking load of wounds in the ileum and colon in comparison with wounds from saline-treated animals. In a further group of animals receiving steroids, paired longitudinal wounds were constructed. One wound of a pair was treated with a local application of transforming growth factor beta (TGF-beta) (5 micrograms per wound) or basic fibroblast growth factor (5 micrograms per wound) in a collagen suspension. The other wound was treated with a collagen suspension alone. Ileal wounds treated with TGF-beta were significantly stronger than collagen-treated controls at 7 days. The steroid-induced impairment of breaking load in intestinal wounds is partially reversed by a local application of TGF-beta in a collagen suspension at the time of surgery.     

Insulinlike growth factor II and transforming growth factor beta regulate collagen expression in human osteoblastlike cells in vitro.

Insulinlike growth factor II (IGF-II) and transforming growth factor beta (TGF-beta) are the most abundant polypeptide growth factors found in human bone matrix and are produced by human bone cells in vitro. IGF-II and TGF-beta 1 increased total protein synthesis, collagenous protein synthesis, and the steady-state level of type I procollagen mRNA in a time-dependent manner in osteoblastlike cells isolated from human bone. Type III procollagen mRNA expression was low in untreated cultures and was not affected by IGF-II or TGF-beta. TGF-beta 1 elevated type I procollagen mRNA rapidly, with the maximal observed change at 10 h. In contrast, procollagen mRNA levels increased more slowly in response to IGF-II and reached a lower maximal level than with TGF-beta, but the response was sustained through 24 h. Collagenous protein synthesis in IGF-II- and TGF-beta-treated cells increased in parallel with increases in procollagen mRNA levels and was higher at 21 h for TGF-beta 1 and at 36 h for IGF-II. The difference in the time course and magnitude of change in type I procollagen mRNA levels in response to IGF-II and TGF-beta 1 suggests that these two growth factors work through distinct mechanisms that provide both a rapid transient response and a later sustained response in bone matrix biosynthetic activity.     

Synergistic effect of transforming growth factor beta and fibroblast growth factor on DNA synthesis in chick growth plate chondrocytes

Transforming growth factor beta and fibroblast growth factor are mitogens for chick growth plate chondrocytes. TGF-beta stimulated a 3.5-fold increase, and FGF a 13.5-fold increase in the rate of thymidine incorporation after a 24 h exposure. TGF-beta and FGF were synergistic in chondrocytes, causing a 73-fold stimulation in thymidine incorporation compared with control. This synergistic response was not dependent upon the simultaneous presence of both mitogens. Sequential exposure of chondrocytes to TGF-beta and FGF in either order reproduced in large part the synergistic interaction observed when both growth factors were present simultaneously. The time required for induction of the subsequent synergistic response was brief and, in the case of TGF-beta, corresponded to the time required for [125I]TGF-beta receptor binding. EGF and PDGF were not mitogenic for chondrocytes, and neither of these factors enhanced the response of the cells to either TGF-beta or FGF. Finally, TGF-beta and FGF did not, either alone or in combination, elevate intracellular cAMP levels. These results emphasize the importance of examining growth factor effects in the context of other growth regulators. Furthermore, this specific and dramatic synergistic stimulation of thymidine incorporation may provide a useful tool in elucidating the mitogenic mechanism of the individual growth factors.     

In vivo induction of bone by recombinant human transforming growth factor beta 1.

A single application of recombinant human transforming growth factor beta 1 (rhTGF-beta 1) adjacent to cartilage was found to induce bone formation in rabbit ear full-thickness skin wounds. At doses that optimally promote soft tissue healing, 25-100 ng rhTGF-beta 1 per wound caused osseous tissue formation starting 21 days after wounding to reach a peak incidence and area of bone formation at day 42. Bone formation was followed by active remodeling, resulting in lower incidence and area of bone formation at days 56 and 70. The early phase of bone formation was located overlying the cartilage and involved perichondrial cells that appeared to differentiate directly into osteoblasts forming bone matrix without a cartilage precursor. Cartilage was replaced with bone at later time points. rhTGF-beta 1 was able to increase the ratio of osteoblasts to osteoclasts lining the trabecular surface of bone and thus increase the net amount of bone formation. The present studies suggest a potential therapeutic role for rhTGF-beta 1 in hard tissue repair.     

成纤维细胞生长因子受体1在胰腺癌细胞中的表达和调控

目的 研究成纤维细胞生长因子受体1(FGFR1)蛋白和mRNA在胰腺癌细胞系中的表达和调控机制.方法 采用Western免疫印迹实验、Northern印迹分析和RT-PCR检测FGFR1在胰腺癌细胞中的表达.使用外源性生长因子刺激细胞并使用激酶抑制剂阻断细胞内信号转导通路,观察FGFR1蛋白和mRNA在胰腺癌细胞中表达的变化情况.结果 FGFR1蛋白和mRNA在胰腺癌细胞系中均有不同程度的表达.生长因子刺激可上调FGFR1蛋白和mRNA的表达水平.其中IGF-1、EGF和FGF2显著增加Mia PaCa-2细胞FGFR1的表达,EGF和FGF2显著增加PANC-1细胞FGFR1的表达(P＜0.05).FGF2对FGFR1表达的调节具有时间依赖性.ERK1/2抑制剂UO126和p38 MAPK抑制剂SB203580降低了PANC-1细胞中FGFR1的蛋白和mRNA的表达水平. 结论生长因子可上调FGFR1在胰腺癌细胞中的表达水平,MAPK信号转导通路中的ERK1/2和p38 MAPK亚通路参与FGFR1表达的调节.     

Localization of transforming growth factors beta1 and beta2 and epidermal growth factor in IgA nephropathy.

OBJECTIVE: The localization of transforming growth factor (TGF)-beta1. TGF-beta2 and epidermal growth factor (EGF) was investigated in IgA nephropathy, and was compared with the severity of histological damage (including tubulointerstitial lesions). MATERIALS AND METHODS: The enzyme antibody method was used to stain paraffin-embedded sections of renal tissue from 42 patients with IgA nephropathy (19 males and 23 females). Results: There was a significant correlation between glomerular positivity for TGF-beta1 and TGF-beta2 and the severity of histological damage. There was also a significant correlation between positivity for TGF-beta1 and TGF-beta2 in the tubular epithelium and tubulointerstitial lesions. In contrast, there was no relationship between glomerular positivity for EGF and histological damage, although there was a significant correlation between positivity for EGF in the tubular epithelium and tubulointerstitial lesions. Conclusions: These findings suggest that TGF-beta1 and TGF-beta2 may be important in the progression of IgA nephropathy, and that the distribution of EGF may also be a useful marker for the progression of renal damage, including tubulointerstitial lesions.     

Inhibition of lymphocyte function by glioblastoma-derived transforming growth factor beta 2

Human glioblastoma cells secrete an inhibitory factor termed "glioblastoma-derived T-cell suppressor factor" (G-TsF). A member of the transforming growth factor beta (TGF beta) family, G-TsF is identical to TGF beta 2. The present study investigated the effect of G-TsF/TGF beta 2 on the proliferative and cytotoxic properties of tumor-infiltrating lymphocytes (TIL's) isolated from malignant gliomas after expansion in vitro with interleukin-2 (IL-2). The results demonstrate that the IL-2 (5 to 20 U/ml)-dependent proliferative response of glioma-derived TIL's was inhibited 70% to 85% by G-TsF/TGF beta 2 and that the inhibitory effect could be reduced by using increasing concentrations of IL-2 (100 to 200 U/ml). Tumor necrosis factor alpha (TNF alpha) enhanced the IL-2-dependent proliferation of TIL's cultured in low concentrations of IL-2 (10 U/ml); however, neither TNF alpha nor interferon gamma was able to reduce the inhibitory effect of TGF beta 2 on TIL proliferation. In addition, TGF beta 2 suppressed 60% to 100% the cytotoxic response of glioma-derived TIL's against several tumor targets, including autologous glioma cells, and the suppressive effect was shown to be reduced by increasing concentrations of IL-2.