与Smad4相互作用的新蛋白HP的鉴定及特性研究
摘要
转化生长因子-β(transforming growth factor-β,TGF-β)信号通路在细胞生长、分化、迁移和凋亡过程中具有很重要的作用。在此信号通路中,TGF-β先通过其受体磷酸化调节型Smad(receptor regulated Smad,R-Smad),然后磷酸化的R-Smad与通用调节Smad(common-mediator Smad,co-Smad)形成复合物,复合物入核直接参与目的基因的调节,从而调控细胞的生长和分化。然而,在TGF-β信号途径中介导细胞生长抑制的Smad调节因子的研究甚少。
Smad4在TGF-β信号转导途径中处于中枢地位。Smad4对恶性肿瘤的发生、发展及转移有重要作用。Smad4(260-514aa)能诱导大规模染色质结构伸展。这一区域可能通过与其它蛋白质的相互作用来发挥Smad4的转录调节功能。本研究以Smad4(260-514aa)为诱饵,用酵母双杂交技术从人乳腺cDNA文库中筛选与Smad4(260-514aa)相互作用的蛋白,分离到了与Smad4相互作用的未知蛋白质—HP,并探讨了HP基因及其表达蛋白的性质、HP的生物学功能、HP参与调节TGF-β信号通路的可能作用模式。
HP基因及其表达蛋白的性质的初探。将分离得到的新基因进行克隆,成功得到了真核表达重组质粒pcDNA3-FLAG-HP。生物信息学分析发现HP含有21个补体调控蛋白(complement control protein,CCP)和两个半CCP序列元件。Western Blot分析显示HP在细胞裂解物及细胞培养上清液中都有表达。免疫荧光染色技术,通过对HP载体在转染MCF-7细胞后不同时间点表达过程的观察,验证了HP是分泌蛋白。通过Edman降解,最终分析得到HP N端前16个氨基酸的信号肽序列。
体内外试验验证了HP与Smad4存在相互作用。应用免疫共沉淀试验检测,结果显示HP与Smad4在哺乳动物细胞内存在结合。通过GST沉淀检测,结果显示HP与Smad4在体外存在相互作用。上述结果验证了HP与Smad4在体内外存在相互作用。
检测了HP的转录调节活性。HP以及HP的缺失突变体能以不依赖TGF-β的方式,不同程度地抑制3TP荧光素酶报告基因的转录。293T细胞中,HP增强TGF-β应答元件的c-Myc报告基因转录活性,同时抑制了PAI-1、P21报告基因转录活性。而在MCF-7细胞中,PAI-1、P21报告基因转录活性升高,c-Myc报告基因转录活性降低。这预示了HP调节TGF-β应答元件的报告基因转录活性具有细胞特异性。HP能以雌激素不依赖的方式抑制雌激素反应元件(estrogen response element,ERE)的转录活性,该结果表明HP参与调节的TGF-β/Smads信号转导与ER信号通路存在串话调节。将RNA干扰(RNA interference,RNAi)实验与活性实验相结合,发现抑制HP的表达后,MCF-7细胞中HP增强TGF-β应答元件的报告基因转录活性的功能也受到抑制。
检测了HP的蛋白表达调节活性。通过G418抗性筛选后得到HP稳定转染细胞株HP-MCF-7。Western Blot检测,HP过量表达激活(周期依赖激酶抑制剂)P21、P15、P27、纤溶酶原激活剂抑制因子-1(plasminogen activator inhibitor-1,PAI-1)、Smad4、BRCA1的表达;同时抑制促进细胞生长的c-Myc、c-jun、Rb、Bcl-2、c-fos的蛋白表达。此结果说明了HP具有蛋白表达调节活性,并且HP有可能作为一种抑癌蛋白,协同增强抑癌蛋白的表达,同时拮抗癌蛋白标志分子的表达。
研究了HP的细胞学功能。流式细胞仪分析结果得出HP的高表达,使细胞生长阻滞在G2/M期。用结晶紫细胞生长曲线测定的方法检测表明MCF-7-HP的生长速度显著慢于MCF-7-149细胞。HP蛋白分泌表达24h,48h,72h都能不同程度地抑制乳腺癌细胞、肺癌细胞、肝癌细胞的生长;同时对正常人乳腺细胞、正常肺、肝细胞、正常成纤维细胞的生长以及SV40转化的人胚胎肾细胞抑制不明显。利用软琼脂实验检测HP使肿瘤细胞集落形成能力降低。
探讨HP参与调节TGF-β信号通路的可能机理。收集用TβRⅡ受体抑制剂SB431542处理的稳定转染细胞株,Western Blot分析,不同剂量SB431542处理的细胞TGF-β下游基因的蛋白表达与对照相比没有明显差异。结果表明,分泌蛋白HP不是直接与膜上Ⅱ型受体结合,有可能是与胞外配体结合,以活化配体调节胞内下游信号的转导。
上述研究结果表明,HP可能是TGF-β信号通路的一个新型调控因子,通过与Smad4相互作用而参与TGF-β信号通路的调控,进一步的深入研究将有助于了解TGF-β信号通路的调控机制。
The transforming growth factor-β(TGF-β) signaling plays an important role in cellular differentiation, apoptosis, and proliferation. Smad2, Smad3 and Smad4 proteins are considered to be key mediators of TGF-βsignaling. However, identifications of Smad partners mediating TGF-βsignaling remain rarely reported.
Smad4 seems to have a center status in TGF-βsignal transduction pathway. A great quantity of researches indicated that Smad4 plays an important part in the generation, development, and metastasis of malignant tumor. It has been founded that Smad4 induces a large scale of chromatin unfolding by targeting itself to an amplified lac operator-containing chromosome region in mammalian cells, and mapped the unfolding activity at the subdomains within the transactivition domain of Smad4 (260-514aa). By yeast two-hybrid screen, HP, a novel Smad4-interacting protein was isolated from a human mammary library with Smad4 (260-514aa) as bait.
To further study the structure, cell location and biological function of HP, the corresponding gene was cloned to construct an eukaryotic expression recombinant plasmid pcDNA3-FLAG-HP. The fact that HP was located in the mammalian cells as a secreted protein was found when the cell lysis and the cell culture supernatant were collected for Western Blot analysis. Furthermore, the recombinant plasmid pcDNA3-HP-enhanced green fluorescent protein (EGFP), expressing HP and EGFP tag, was transfected into MCF-7 cells. The secreting expression process of HP was observed under the fluorescence microscope at different time after transfection.
When studying functional activities, it was showed that HP and the deletants can inhibit the transcription of TGF-βresponsive 3TP-luciferase (luc) reporter gene in a TGF-β-independent manner to some extent. The result also showed that HP enhanced c-Myc reporter gene transcriptional activity, meanwhile inhibited PAI-1, P21 transcriptional activity in 293T cells, while the results were opposite in MCF-7 cells with distinct mediation in different cells. In the luc system, the results demonstrated that HP inhibited ERE-Luciferase transcriptional activity, indicating that HP also medicated ER signal transduction by "cross-talk". Subsequently, the expression vectors of HP-small interfering RNAs (siRNAs) were constructed and confirmed by DNA sequencing. Western Blot analysis showed that HP-siRNAs could effectively inhibit the expression of HP gene. It was also discovered that the ability of HP to enhance the transcription of P21 reporter gene was inhibited when MCF-7 cells were co-transfected with HP-siRNAs.
Biological functional of HP in cells were further studied. Stable FLAG-HP transfected MCF-7 cell was obtained after G418 resist screening. It was detected by Western Blot that the expressions of P21, P15, P27, plasminogen activator inhibitor-1 (PAI-1), Smad4 and BRCA1 were enhanced and the expressions of c-Myc, c-jun, Rb, Bcl-2 and c-fos proteins were decreased. Meanwhile, MCF-7 cell growth retardation at G2/M was observed by cell flow meter analysis. With viola crystallina assay, HP stable taransfected cells and control were detected to study the influence of HP overexpression on cell growth, and the results showed that MCF-7-HP growed significantly slower than MCF-7-149. Moreover, the conditional medium of HP-MCF-7 and 149-MCF-7 was collected to study the influence of secreted HP protein on cell growth. The results indicated that one, two and three days cultured conditional medium can inhibit the growth of breast cancer, lung cancer, hepatoma cancer cells at distinct levels; while the inhibition was not obvious in normal cells, including normal breast cells, lung cells, hepatoma cells, fibroblastic cells and SV40 transfromed 293T cells.
TβRⅡinhibitor SB431542 was added to explore whether HP contact with TβR in signal transduction pathway. No obvious difference was obtained in Western Blot analysises at different times and different doses of SB431542 treated protein expression, including P21、PAI-1、c-Myc. The result indicated that secreted HP protein did not contact with TβRⅡbut possibly with ligands to activate TβRⅠto mediate the signal pathway.
Implication: these results identified a new regulating factor, HP, which plays a role in TGF-βsignaling pathway through interaction with Smad4. Further study will be helpful to understand its specific regulating mechanism in TGF-βsignaling pathway.
Smad4在TGF-β信号转导途径中处于中枢地位。Smad4对恶性肿瘤的发生、发展及转移有重要作用。Smad4(260-514aa)能诱导大规模染色质结构伸展。这一区域可能通过与其它蛋白质的相互作用来发挥Smad4的转录调节功能。本研究以Smad4(260-514aa)为诱饵,用酵母双杂交技术从人乳腺cDNA文库中筛选与Smad4(260-514aa)相互作用的蛋白,分离到了与Smad4相互作用的未知蛋白质—HP,并探讨了HP基因及其表达蛋白的性质、HP的生物学功能、HP参与调节TGF-β信号通路的可能作用模式。
HP基因及其表达蛋白的性质的初探。将分离得到的新基因进行克隆,成功得到了真核表达重组质粒pcDNA3-FLAG-HP。生物信息学分析发现HP含有21个补体调控蛋白(complement control protein,CCP)和两个半CCP序列元件。Western Blot分析显示HP在细胞裂解物及细胞培养上清液中都有表达。免疫荧光染色技术,通过对HP载体在转染MCF-7细胞后不同时间点表达过程的观察,验证了HP是分泌蛋白。通过Edman降解,最终分析得到HP N端前16个氨基酸的信号肽序列。
体内外试验验证了HP与Smad4存在相互作用。应用免疫共沉淀试验检测,结果显示HP与Smad4在哺乳动物细胞内存在结合。通过GST沉淀检测,结果显示HP与Smad4在体外存在相互作用。上述结果验证了HP与Smad4在体内外存在相互作用。
检测了HP的转录调节活性。HP以及HP的缺失突变体能以不依赖TGF-β的方式,不同程度地抑制3TP荧光素酶报告基因的转录。293T细胞中,HP增强TGF-β应答元件的c-Myc报告基因转录活性,同时抑制了PAI-1、P21报告基因转录活性。而在MCF-7细胞中,PAI-1、P21报告基因转录活性升高,c-Myc报告基因转录活性降低。这预示了HP调节TGF-β应答元件的报告基因转录活性具有细胞特异性。HP能以雌激素不依赖的方式抑制雌激素反应元件(estrogen response element,ERE)的转录活性,该结果表明HP参与调节的TGF-β/Smads信号转导与ER信号通路存在串话调节。将RNA干扰(RNA interference,RNAi)实验与活性实验相结合,发现抑制HP的表达后,MCF-7细胞中HP增强TGF-β应答元件的报告基因转录活性的功能也受到抑制。
检测了HP的蛋白表达调节活性。通过G418抗性筛选后得到HP稳定转染细胞株HP-MCF-7。Western Blot检测,HP过量表达激活(周期依赖激酶抑制剂)P21、P15、P27、纤溶酶原激活剂抑制因子-1(plasminogen activator inhibitor-1,PAI-1)、Smad4、BRCA1的表达;同时抑制促进细胞生长的c-Myc、c-jun、Rb、Bcl-2、c-fos的蛋白表达。此结果说明了HP具有蛋白表达调节活性,并且HP有可能作为一种抑癌蛋白,协同增强抑癌蛋白的表达,同时拮抗癌蛋白标志分子的表达。
研究了HP的细胞学功能。流式细胞仪分析结果得出HP的高表达,使细胞生长阻滞在G2/M期。用结晶紫细胞生长曲线测定的方法检测表明MCF-7-HP的生长速度显著慢于MCF-7-149细胞。HP蛋白分泌表达24h,48h,72h都能不同程度地抑制乳腺癌细胞、肺癌细胞、肝癌细胞的生长;同时对正常人乳腺细胞、正常肺、肝细胞、正常成纤维细胞的生长以及SV40转化的人胚胎肾细胞抑制不明显。利用软琼脂实验检测HP使肿瘤细胞集落形成能力降低。
探讨HP参与调节TGF-β信号通路的可能机理。收集用TβRⅡ受体抑制剂SB431542处理的稳定转染细胞株,Western Blot分析,不同剂量SB431542处理的细胞TGF-β下游基因的蛋白表达与对照相比没有明显差异。结果表明,分泌蛋白HP不是直接与膜上Ⅱ型受体结合,有可能是与胞外配体结合,以活化配体调节胞内下游信号的转导。
上述研究结果表明,HP可能是TGF-β信号通路的一个新型调控因子,通过与Smad4相互作用而参与TGF-β信号通路的调控,进一步的深入研究将有助于了解TGF-β信号通路的调控机制。
The transforming growth factor-β(TGF-β) signaling plays an important role in cellular differentiation, apoptosis, and proliferation. Smad2, Smad3 and Smad4 proteins are considered to be key mediators of TGF-βsignaling. However, identifications of Smad partners mediating TGF-βsignaling remain rarely reported.
Smad4 seems to have a center status in TGF-βsignal transduction pathway. A great quantity of researches indicated that Smad4 plays an important part in the generation, development, and metastasis of malignant tumor. It has been founded that Smad4 induces a large scale of chromatin unfolding by targeting itself to an amplified lac operator-containing chromosome region in mammalian cells, and mapped the unfolding activity at the subdomains within the transactivition domain of Smad4 (260-514aa). By yeast two-hybrid screen, HP, a novel Smad4-interacting protein was isolated from a human mammary library with Smad4 (260-514aa) as bait.
To further study the structure, cell location and biological function of HP, the corresponding gene was cloned to construct an eukaryotic expression recombinant plasmid pcDNA3-FLAG-HP. The fact that HP was located in the mammalian cells as a secreted protein was found when the cell lysis and the cell culture supernatant were collected for Western Blot analysis. Furthermore, the recombinant plasmid pcDNA3-HP-enhanced green fluorescent protein (EGFP), expressing HP and EGFP tag, was transfected into MCF-7 cells. The secreting expression process of HP was observed under the fluorescence microscope at different time after transfection.
When studying functional activities, it was showed that HP and the deletants can inhibit the transcription of TGF-βresponsive 3TP-luciferase (luc) reporter gene in a TGF-β-independent manner to some extent. The result also showed that HP enhanced c-Myc reporter gene transcriptional activity, meanwhile inhibited PAI-1, P21 transcriptional activity in 293T cells, while the results were opposite in MCF-7 cells with distinct mediation in different cells. In the luc system, the results demonstrated that HP inhibited ERE-Luciferase transcriptional activity, indicating that HP also medicated ER signal transduction by "cross-talk". Subsequently, the expression vectors of HP-small interfering RNAs (siRNAs) were constructed and confirmed by DNA sequencing. Western Blot analysis showed that HP-siRNAs could effectively inhibit the expression of HP gene. It was also discovered that the ability of HP to enhance the transcription of P21 reporter gene was inhibited when MCF-7 cells were co-transfected with HP-siRNAs.
Biological functional of HP in cells were further studied. Stable FLAG-HP transfected MCF-7 cell was obtained after G418 resist screening. It was detected by Western Blot that the expressions of P21, P15, P27, plasminogen activator inhibitor-1 (PAI-1), Smad4 and BRCA1 were enhanced and the expressions of c-Myc, c-jun, Rb, Bcl-2 and c-fos proteins were decreased. Meanwhile, MCF-7 cell growth retardation at G2/M was observed by cell flow meter analysis. With viola crystallina assay, HP stable taransfected cells and control were detected to study the influence of HP overexpression on cell growth, and the results showed that MCF-7-HP growed significantly slower than MCF-7-149. Moreover, the conditional medium of HP-MCF-7 and 149-MCF-7 was collected to study the influence of secreted HP protein on cell growth. The results indicated that one, two and three days cultured conditional medium can inhibit the growth of breast cancer, lung cancer, hepatoma cancer cells at distinct levels; while the inhibition was not obvious in normal cells, including normal breast cells, lung cells, hepatoma cells, fibroblastic cells and SV40 transfromed 293T cells.
TβRⅡinhibitor SB431542 was added to explore whether HP contact with TβR in signal transduction pathway. No obvious difference was obtained in Western Blot analysises at different times and different doses of SB431542 treated protein expression, including P21、PAI-1、c-Myc. The result indicated that secreted HP protein did not contact with TβRⅡbut possibly with ligands to activate TβRⅠto mediate the signal pathway.
Implication: these results identified a new regulating factor, HP, which plays a role in TGF-βsignaling pathway through interaction with Smad4. Further study will be helpful to understand its specific regulating mechanism in TGF-βsignaling pathway.
引文
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