Hedgehog信号通路蛋白在肝内胆管细胞癌中的表达

目的 研究Hedgehog(Hh)信号通路相关蛋白(Gli 1,Ptch-1和Shh)在肝内胆管细胞癌(intrahepatic cholangio carcinoma,ICC)中的表达及意义.通过对ICC和癌旁组织中Hh通路相关蛋白(Gli 1、Ptch-1和Shh)的表达进行检测,探讨其与ICC临床病理特征之间的关系及意义.方法 免疫组化法对43例ICC组织及癌旁组织进行Gli 1、Ptch-1和Shh蛋白的检测;Real-time RT-PCR检测12例ICC组织和癌旁组织中mRNA的表达,并与患者临床病理特征进行相关性分析.结果 43例ICC组织中,Gli 1、Ptch-1和Shh阳性表达率分别为74.4%(32/43)、69.8% (30/43),和62.8%(27/43),与癌旁组织表达比较差异具有统计学意义(P值分别为0.003、0.006和0.008);三者的表达与肿瘤的大小有关(P值分别为0.039、0.044和0.047);三者的表达与肿瘤的分化程度相关(P值分别为0.026,0.039和0.049);三者的表达与淋巴结转移具有相关性(P均<0.05).Real-time PCR结果显示,12例ICC组织中均有Gli 1、Ptch-1和Shh的表达,且基因表达水平均高于癌旁正常组织(P均=0.001).结论 Hedgehog信号通路的相关蛋白在ICC组织中高表达,其异常激活可能与肝内胆管细胞癌发生密切相关.Gli 1、Ptch-1和Shh的表达可能与肿瘤的生长和转移密切相关.     

Hedgehog signaling is involved in differentiation of normal colonic tissue rather than in tumor proliferation

The Hedgehog (Hh) pathway is a main regulation cascade in embryonic differentiation. It is also present in adult tissues and unusual expression has been associated with formation of benign and malignant lesions. We examined the presence of the Hedgehog pathway in normal and pathological human colon tissue. Components investigated include Sonic (Shh), Indian (Ihh), and Desert Hedgehog (Dhh), Gli1, Gli2, Gli3, and Patched (Ptch). Pathological tissue samples comprised 23 benign and 20 malignant lesions of human colon. The influence of the Hedgehog pathway on differentiation and proliferation has been investigated by analyzing the effect of the pathway inhibitor Cyclopamine on human colon cancer cell lines HT29 and CaCo2. In normal colon, we detected expression of Shh and Dhh within the lining epithelium and Patched, Gli1, and Gli2 along the whole crypts. Within all benign lesions, positive staining of Shh, Dhh, Gli1, Gli2, and Ptch was detected. Expression of Shh and Dhh was restricted to single cell aggregates. Malignant lesions also displayed focal staining pattern for Shh and Dhh but to a much lesser extent. We conclude that Hedgehog signaling is involved rather in constant differentiation and renewing of the colonic lining epithelium than in cancer formation, growth, or proliferation.     

阻断Sonic Hedgehog信号对不同人肝癌细胞生长的影响

 目的: 研究阻断Sonic Hedgehog (Shh)信号对不同人肝癌细胞生长的影响,探讨阻断Shh信号抑制肝癌细胞生长的机制。方法: RT-PCR法检测Shh信号分子在3株人肝癌细胞(BEL-7402、Huh7和HepG2)中的表达,并检测Shh阻断抗体作用后BEL-7402细胞Shh信号效应分子表达变化;MTT法检测人肝癌细胞增殖活性;流式细胞术检测人肝癌细胞凋亡;Western blot 检测凋亡相关蛋白表达。结果: Shh信号分子在3株人肝癌细胞中均有表达,Shh阻断抗体可以下调Shh信号效应分子patched (Ptch)、Gli1和Gli2的表达;Shh阻断抗体可以抑制3株肝癌细胞生长,增加G₀/G₁期细胞,并诱导细胞凋亡;Shh阻断抗体作用后,BEL-7402细胞pro-caspase-3、pro-caspase-8和pro-caspase-9蛋白表达水平下降,cleaved caspase-3、cleaved caspase-8和cleaved caspase-9蛋白表达水平升高。结论: 阻断Shh信号可抑制Shh高表达的人肝癌细胞生长,阻滞细胞周期于G₀/G₁期,并诱导肝癌细胞凋亡。     

Hedgehog signaling regulates proliferation of prostate cancer cells via stathmin1

Hedgehog (Hh) signaling is an essential pathway in embryonic development of prostate. Hh also plays roles in the proliferation of progenitor cells and cancer cells of adult prostate. However, how Hh signaling contributes to carcinogenesis of prostate is poorly understood. Stathmin1 is a microtubule-regulating protein that plays an important role in the assembly and disassembly of the mitotic spindle. Stathmin1 is expressed in normal developing mouse prostate and in prostate cancer. The expression pattern of stathmin1 is similar to that of Shh in prostate development and cancer, suggesting a connection between these two proteins. In this study, we examined the relationship between stathmin1 and Hh signaling. Here, we show that stathmin1 expression is regulated by Hh signaling in prostate cancer cells. Cyclopamine, a specific inhibitor of Hh signaling, reduced the expression of stathmin1 in prostate cancer cells. However, the Shh peptide induced stathmin1 expression. Overexpression of Gli1 further confirmed the relationship. Co-expression of stathmin1 and Patched 1, a receptor for Hh signaling was observed in prostate cancer tissues. Cyclopamine and stathmin1 siRNA both decreased proliferation of prostate cancer cells but did not produce an additive effect, suggesting a common pathway. These results suggest that Hh signaling regulates proliferation of prostate cancer cells by controlling stathmin1 expression.     

Sonic hedgehog stimulates mouse embryonic stem cell proliferation by cooperation of Ca2+/protein kinase C and epidermal growth factor receptor as well as Gli1 activation

Hedgehog signaling has an essential role in the control of stem cell growth in embryonic tissues. Therefore, this study examined the effect of sonic hedgehog (Shh) on the self-renewal of mouse embryonic stem (ES) cells and its related mechanisms. Shh increased DNA synthesis blocked by the inhibition of the smoothened receptor. Shh required Gli1 activation to induce the increases in Notch/Hes-1 and Wnt/beta-catenin. Shh increased the intracellular calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity. We show that the Shh-induced increase in the Gli1 mRNA level requires [Ca(2+)](i) and PKC. Shh increased the phosphorylation of epidermal growth factor receptor (EGFR), which is blocked by the matrix metalloproteinase inhibitor. Subsequently, Shh increased the nuclear factor (NF)-kappaB p65 phosphorylation, which was inhibited by blocking PKC and EGFR tyrosine kinase. Shh also increased the level of the cell cycle regulatory proteins in a dose-dependent manner. However, Shh decreased the levels of the cyclin-dependent kinase inhibitory proteins. The effect of Shh on these proteins was inhibited by blocking PKC, EGFR, and NF-kappaB as well as transfection of Gli1 small interfering RNA (siRNA). Finally, Shh-induced progression of the G1/S-phase was blocked by the inhibition of PKC and EGFR tyrosine kinase. In conclusion, Shh stimulates mouse ES cell proliferation through Gli1 activation as well as Ca(2+)/PKC and EGFR. Disclosure of potential conflicts of interest is found at the end of this article.     

槲芪散可通过激活Hedgehog信号通路抑制大鼠肝癌前病变的形成

目的:探讨槲芪散是否通过调控Hedgehog信号通路抑制大鼠肝癌前病变的机制。方法:采用改良的Solt-Farber二步法复制大鼠肝癌前病变模型,于肝大部切除术后3 d开始给予槲芪散(8 g/kg)灌胃治疗,4周后收集血清和肝脏标本。分别采用HE染色、免疫组织化学、免疫荧光染色、Western blot、实时荧光定量PCR等方法,观察肝脏病理变化,检测肝组织谷胱甘肽S-转移酶π(glutathione S-transferase-π,GST-π)、甲胎蛋白(alpha-fetoprotein,AFP)、OV6、白蛋白(albumin,ALB)、Hedgehog信号分子Shh、Smo、Gli2及下游靶分子cyclin D、cyclin E的表达水平;利用比色法检测血清丙氨酸转氨酶(alanine aminotransferase,ALT)、天冬氨酸转氨酶(aspartate aminotransferase,AST)和γ-谷氨酰转移酶(gamma-glutamyltransferase,GGT)水平;给予Hedgehog信号通路抑制剂环杷明观察槲芪散在体外对肝卵圆细胞的影响。结果:与模型组比较,槲芪散可降低肝癌前病变大鼠血清ALT、AST和GGT水平,减轻肝癌前病变大鼠肝脏的病理变化,降低肝癌前病变标志物GST-π和AFP的表达;槲芪散可促使肝卵圆细胞标志物OV6和肝细胞标志物ALB的表达;槲芪散可激活肝脏Hedgehog信号通路,增加Shh、Smo、Gli2及其下游靶分子cyclin D、cyclin E的表达。结论:中药复方槲芪散可通过激活Hedgehog信号通路促进肝前体细胞增殖和诱导肝前体细胞向肝细胞分化,抑制肝癌前病变的形成,促进肝脏修复。     

Major components of the insulin-like growth factor axis are expressed early in chicken embryogenesis,with IGF binding protein ( IGFBP) -5 expression subject to regulation by Sonic Hedgehog

Using whole-mount in situ hybridisation techniques, we have examined the expression of major components of the insulin-like growth factor (IGF) axis in early development of the chicken embryo, including both IGF-I and -II, the type 1 IGF receptor ( IGFR), and two of the IGF binding proteins, ( IGFBP) -2 and -5. We report that these genes fall into two distinct groups with respect to expression pattern, with IGFBP-2 displaying broad overlap of mRNA expression with IGFR and IGF-I during early development, whereas the expression profile of IGFBP-5 most closely resembled that of IGF-II. Comparison between different stages revealed IGFBP-2 mRNA was detected as early as stage 3, whereas IGFBP-5 was first seen at stage 4. In addition, we detected expression domains of IGFBP-5, and to a lesser extent IGFBP-2, which did not overlap with either IGFR or IGF expression patterns. This could indicate IGF independent actions of the IGFBPs during early embryonic development. A striking observation concerning the expression profiles of both IGF-II and IGFBP-5 at early stages of chick embryogenesis is that both these genes are expressed asymmetrically in a pattern similar to that of Sonic Hedgehog (Shh). Furthermore, using cyclopamine, we have demonstrated that IGFBP-5 expression in the early embryo is regulated by Shh. Taken together, these results describe an important role for the IGF system in the very early stages of the developing chicken embryo, and imply that IGFBP-2 and -5 are fundamental developmental factors, with the latter involved in Shh signalling pathways.     

Core binding factor beta functions in the maintenance of stem cells and orchestrates continuous proliferation and differentiation in mouse incisors

Rodent incisors grow continuously throughout life, and epithelial progenitor cells are supplied from stem cells in the cervical loop. We report that epithelial Runx genes are involved in the maintenance of epithelial stem cells and their subsequent continuous differentiation and therefore growth of the incisors. Core binding factor β (Cbfb) acts as a binding partner for all Runx proteins, and targeted inactivation of this molecule abrogates the activity of all Runx complexes. Mice deficient in epithelial Cbfb produce short incisors and display marked underdevelopment of the cervical loop and suppressed epithelial Fgf9 expression and mesenchymal Fgf3 and Fgf10 expression in the cervical loop. In culture, FGF9 protein rescues these phenotypes. These findings indicate that epithelial Runx functions to maintain epithelial stem cells and that Fgf9 may be a target gene of Runx signaling. Cbfb mutants also lack enamel formation and display downregulated Shh mRNA expression in cells differentiating into ameloblasts. Furthermore, Fgf9 deficiency results in a proximal shift of the Shh expressing cell population and ectopic FGF9 protein suppresses Shh expression. These findings indicate that Shh as well as Fgf9 expression is maintained by Runx/Cbfb but that Fgf9 antagonizes Shh expression. The present results provide the first genetic evidence that Runx/Cbfb genes function in the maintenance of stem cells in developing incisors by activating Fgf signaling loops between the epithelium and mesenchyme. In addition, Runx genes also orchestrate continuous proliferation and differentiation by maintaining the expression of Fgf9 and Shh mRNA.     

Sonic Hedgehog stimulates migration of MCF-7 breast cancer cells through Rac1

As one of the most common tumors in women, breast cancer has drawn considerable interest from investigators and clinicians in recent years. Despite early diagnosis and best therapeutic regimens available, the prognosis of malignant or metastatic breast cancer patients is still not optimistic. Hedgehog signaling, a classical pathway indispensable to embryonic development, participates in the growth of a variety of tumors. In the present study, the effect of Sonic Hedgehog (Shh) on breast cancer cells was investigated. We identified that Shh signal stimulated the migration of MCF-7 breast cancer cells. Smo and Gli1 were involved in Shh-stimulated migration of MCF-7 cells. Activating Smo and Gli1 induced cell migration, which was blocked by their specific antagonists. The effect of Shh signaling on MCF-7 cells was independent of Wnt5a, Dvl2 and Rab35, but directly dependent on Rac1. In conclusion, our study suggested that Shh promotes breast cancer cell migration via Rac1 independently of the non-canonical Wnt signaling pathway, which may represent a rational molecular target for combination medication in breast cancer.     

A direct requirement for Hedgehog signaling for normal specification of all ventral progenitor domains in the presumptive mammalian spinal cord

The hedgehog signaling pathway organizes the developing ventral neural tube by establishing distinct neural progenitor fates along the dorsoventral axis. Smoothened (Smo) is essential for all Hedgehog (Hh) signaling, and genetic inactivation of Smo cells autonomously blocks the ability of cells to transduce the Hh signal. Using a chimeric approach, we examined the behavior of Smo null mutant neural progenitor cells in the developing vertebrate spinal cord, and we show that direct Hh signaling is essential for the specification of all ventral progenitor populations. Further, Hh signaling extends into the dorsal half of the spinal cord including the intermediate Dbx expression domain. Surprisingly, in the absence of Sonic hedgehog (Shh), we observe the presence of a Smo-dependent Hh signaling activity operating in the ventral half of the spinal cord that most likely reflects Indian hedgehog (Ihh) signaling originating from the underlying gut endoderm. Comparative studies of Shh, Smo, and Gli3 single and compound mutants reveal that Hh signaling acts in part to specify neural cell identity by counteracting the repressive action of Gli3 on p0, p1, p2, and pMN formation. However, whereas these cell identities are restored in Gli3/Smo compound mutants, correct stratification of the rescued ventral cell types is lost. Thus, Hh signaling is essential for organizing ventral cell pattern, possibly through the control of differential cell affinities.     

转化生长因子β1诱导心肌成纤维细胞转分化过程中Hedgehog信号通路的作用机制

背景:近年来越来越多的研究表明Hedghog信号通路异常活化广泛参与全身多组织器官疾病的损伤修复过程,但其在心肌纤维化中的相关作用尚不明确.目的:研究阻断Hedgehog-Gli信号通路对转化生长因子β1诱导的心肌成纤维细胞上皮间质转分化过程的影响.方法:①动物实验:左冠状动脉结扎法建立小鼠心肌梗死模型,苏木精-伊红和...     

Levels of Gli3 repressor correlate with Bmp4 expression and apoptosis during limb development.

Removal of the posterior wing bud leads to massive apoptosis of the remaining anterior wing bud mesoderm. We show here that this finding correlates with an increase in the level of the repressor form of the Gli3 protein, due to the absence of the Sonic hedgehog (Shh) protein signaling. Therefore, we used the anterior wing bud mesoderm as a model system to analyze the relationship between the repressor form of Gli3 and apoptosis in the developing limb. With increased Gli3R levels, we demonstrate a concomitant increase in Bmp4 expression and signaling in the anterior mesoderm deprived of Shh signaling. Several experimental approaches show that the apoptosis can be prevented by exogenous Noggin, indicating that Bmp signaling mediates it. The analysis of Bmp4 expression in several mouse and chick mutations with defects in either expression or processing of Gli3 indicates a correlation between the level of the repressor form of Gli3 and Bmp4 expression in the distal mesoderm. Our analysis adds new insights into the way Shh differentially controls the processing of Gli3 and how, subsequently, BMP4 expression may mediate cell survival or cell death in the developing limb bud in a position-dependent manner.