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Title: | 第一部分:表皮生長因子於皮膚角質細胞誘導Blimp-1表現及細胞功能之分子機制探討。 第二部分:探討脾酪胺酸蛋白激酶調控紅血球前驅細胞存活及分化之角色。 Part I: Molecular mechanisms for EGF-induced Blimp-1 expression and cellular functions in keratinocytes. Part II: The role of spleen tyrosine kinase in erythroblast survival and differentiation. |
Authors: | Hua-Ching Chang 張華景 |
Advisor: | 林琬琬 |
Keyword: | 第一部分:轉錄抑制因子,表皮生長因子,皮膚角質細胞,第二部分:脾酪胺酸蛋白激?,紅血球生成,紅血球生成素,顆粒單核球群落刺激生長因子, Part I: Blimp-1,EGF,keratinocyte,Part II: Syk,Erythropoiesis,EPO,GM-CSF, |
Publication Year : | 2015 |
Degree: | 碩士 |
Abstract: | 第一部分: Blimp-1 是一轉錄抑制因子,其在調控眾多免疫細胞的發育及功能上扮演關鍵性的角色,近來研究指出,Blimp-1也會表現於皮膚表皮層中,進行調控皮膚表皮的終端角質分化及抑制自發性皮膚發炎的產生。另一方面,表皮生長因子受器 (EGFR)能夠調控許多皮膚角質細胞重要之功能,諸如生長、移行、分化及免疫功能,藉以維持皮膚的生理恆定。截至今日,關於Blimp-1在皮膚表皮層表現的調控機制及其與EGFR之間的相互關聯性的了解仍相當有限,在目前的研究中,我們利用初級培養人類皮膚角質細胞及皮膚角質細胞株HaCaT作為主要研究材料,用以探討Blimp-1是否會受到表皮生長因子受器的調控,並同時參與表皮生長因子在皮膚角質細胞中所執行的功能。我們發現,表皮生長因子受器配體 (ligands)同時可以增加Blimp-1在人類皮膚角質細胞中的蛋白質及訊息mRNA (mRNA)的表現量,而此現象能夠被表皮生長因子受器抑制劑及基因減弱(Knockdown)的方式所抑制,而表皮生長因子所刺激的Blimp-1增加現象,須依靠表皮生長因子受器下游的PKC、p38及ERK訊息傳遞路徑。此外,Blimp-1的穩定性主要受到蛋白酶體(proteasome)降解系統的控制,輔以溶體(lysosome)降解系統適度的參與,然而,溶體抑制劑bafilomycin A1卻同時可以藉由間接活化表皮生長因子受器,從轉錄階層的調控來提升Blimp-1的表現。在功能上,利用基因減弱模式抑制Blimp-1在皮膚角質細胞的表現,可增加表皮生長因子所媒介的第八型介白素(IL-8)、趨化素CXCL5、及第六型介白素(IL-6)的製造,逆轉表皮生長因子受器活化時抑制的皮膚分化指標Keratin 10,及促進表皮生長因子所誘導的細胞移行能力。另外,我們發現表皮生長因子所調控的Blimp-1增加現象,同樣會發生在皮膚相關的鱗狀上皮細胞癌細胞株A431、Cal-27與TW2.6細胞中,但不會產生在初級培養小鼠皮膚角質細胞,及數種惡性腫瘤細胞株中,如肺癌細胞A549、大腸直腸癌細胞HCT116、乳癌細胞MCF7及肝細胞癌細胞HepG2。結論是,我們目前的研究結果,深入性地瞭解表皮生長因子調控Blimp-1表現的機制,以及Blimp-1在人類皮膚角質細胞中,扮演著負調控表皮生長因子在發炎、分化及移形上的重要角色。 第二部分: 紅血球為無細胞核的細胞,其主要功能在於運送氧氣至各組織,而紅血球生成(Erythropoiesis)是一嚴密調控紅血球自前驅細胞分化至成熟紅血球的過程,許多生長因子、賀爾蒙及細胞激素皆參與調控紅血球生成的過程,而最主要的因子包含紅血球生成素(EPO)及顆粒單核球群落刺激生長因子(GM-CSF),儘管JAK2/STAT5訊息傳遞路徑被認為是EPO及GM-CSF受器的主要起始者,非受器型脾酪胺酸激酶(Syk)也曾被報導參與這兩種受器的下游訊息傳遞,然而,Syk在紅血球前驅細胞的確切生理角色仍屬未知。在本篇研究,我們將探討Syk在EPO及GM-CSF調控紅血球生成時的角色,並且探究是否紅血球參與調控乙型轉化生長因子(TGF-β)的作用,已知TGF-β在抑制紅血球母細胞的增生之餘,可以同時促進其分化。利用人類紅血球母細胞細胞株TF-1,我們發現無論細胞是否處於EPO或GM-CSF的刺激下,Syk的專一性抑制劑R406均能降低細胞存活率,並且與TGF-β具加乘性作用,而R406抑制細胞存活率的機制,源自於抑制細胞週期的增生作用,及增加非凋亡蛋白酶依賴性的細胞凋亡作用(caspase-independent apoptosis),而出乎意料地,Src-家族酪胺酸激酶(SFKs)並未參與Syk在TF-1細胞中調控細胞存活的作用。在訊息傳遞路徑分析上,Syk分別影響EPO受器下游的STAT5活化及GM-CSF下游的Akt磷酸化,並參與調控兩種受器刺激的ERK活性,相反地,TGF-β則不會影響EPO及GM-CSF受器的訊息傳遞。此外,R406會抑制EPO所誘導的紅血球分化標的丙型血紅素(Hbγ)的表現量,而TGF-β則會促進Hbγ的產生。整體而言,Syk是一新發現的EPO及GM-CSF受器的訊息傳遞路徑上游分子,參與調控紅血球生成過程中,促進紅血球母細胞增生、存活及分化的功能,我們目前的發現可以用以解釋在接受Syk抑制劑fostamatinib的臨床試驗患者中,造成貧血副作用的機制。 Part I: B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor, and plays a crucial role in the regulation of development and function of various immune cells. Recent studies indicate that Blimp-1 is also expressed in epidermis of skin, and it could modulate the terminal differentiation of skin epidermis and suppress spontaneous skin inflammation. On the other hand, the epidermal growth factor receptor (EGFR) maintains skin homeostasis through regulation of several pivotal functions of keratinocytes, including proliferation, migration, differentiation, and immune function. Currently, it is poor understanding about the regulation of Blimp-1 expression and the crosstalk between the EGFR and Blimp-1 in human epidermal keratinocytes. In current study, we used primary human keratinocytes and keratinocyte cell line HaCaT to investigate whether Blimp-1 is under regulation of EGFR and involves in EGFR-mediated function in keratinocytes. We found EGFR ligands could upregulate the Blimp-1 protein expression and mRNA level in human keratinocytes, and this effect could be diminished by EGFR inhibitor or knockdown. The upregulation of Blimp-1 by EGF stimulation was dependent on downstream PKC, p38, and ERK signaling pathways. Additionally, the stability of Blimp-1 was majorly under the control of the proteasome degradation system, and moderately of lysosome degradation system. Nevertheless, lysosome inhibitor bafilomycin A1 could also enhance the Blimp-1 expression via transcription level modulation indirectly via EGFR activation. Functionally, Blimp-1 knockdown in keratinocytes enhanced the EGFR-mediated interleukin-8 (IL-8), chemokine (C-X-C motif) ligand 5 (CXCL5) and IL-6 productions, reversed the differentiation marker keratin 10 suppression by EGFR activation, and promoted the migration ability triggered by EGF. Furthermore, we found the phenomenon of EGF-induced Blimp-1 upregulation is similar in selected skin-related squamous cell carcinomas (A431, Cal-27, and TW2.6), but not present in primary mouse keratinocytes, human lung cancer A549, colorectal cancer HCT116, breast cancer MCF7 or hepatocarcinoma HepG2. In conclusion, our findings provide the insight into the EGFR-mediated Blimp-1 expression and the importance of negative regulatory role of Blimp-1 on EGFR function in inflammation, differentiation, and migration of human keratinocytes. Part II: Erythrocytes are anucleate cells that function to transport oxygen to the tissues, and erythropoiesis is a tightly regulated process of differentiation from erythroid progenitor cells to mature erythrocytes. Several growth factors, hormones, and cytokines are involved in erythropoiesis, and the principal factors regulating this process include erythropoietin (EPO) and granulocyte macrophage colony-stimulating factor (GM-CSF). Although JAK2/STAT5 signaling is recognized the major initiator of EPO and GM-CSF receptor signaling, non-receptor spleen tyrosine kinase (Syk) was also reported to be the downstream signaling molecule for both receptors. However, the definite biological function of Syk in erythroid progenitor cells remains unclear. In this study, we investigate the role of Syk in the biological actions of EPO and GM-CSF in erythropoiesis, and also explore whether Syk is involved to modulate the effect of transforming growth factor beta (TGF-β), which can inhibit proliferation but potentiate differentiation of erythroblasts. In human erythroblast cell line TF-1, we found Syk specific inhibitor R406 could decrease cell viability either in the absence or presence of EPO and GM-CSF, and with additive effect with TGF-β. Such inhibitory effect of R406 on cell viability results from reduced cell cycle progression for proliferation and increased cell apoptosis through a caspase-independent mechanism. Unexpectedly, Src family kinases (SFKs) are not involved in the viability control by Syk in TF-1 cells. Signaling studies showed that Syk is required for STAT5 activation induced by EPO, Akt phosphorylation triggered by GM-CSF, and ERK activity stimulated by both EPO and GM-CSF. In contrast, TGF-β does not affect the signaling pathways induced by EPO and GM-CSF. Furthermore, EPO-induced Hbγ expression, an index of erythroid differentiation, was reduced by R406 but enhanced by TGF-β. Taken together, Syk is a novel upstream signaling molecule of EPO and GM-CSF receptors, and involve in modulating erythropoiesis by promoting erythroblast proliferation, survival and differentiation. Our findings can explain the anemia adverse effect that is observed in clinical trial patients receiving Syk inhibitor fostamatinib. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18025 |
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Appears in Collections: | 藥理學科所 |
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