Establishment and characterization of dental epithelial cell lines from human HERS/ERM and dental pulp

김지혜 서울대학교 대학원 2017 국내석사

Stem cells can differentiate into various cell types and develop into any tissue. Adult stem cells are present in very small populations in each tissue of the body and exist as undifferentiated cells. When tissue damage occurs, stem cells rapidly differentiate and regenerate the tissue. Despite their limited differentiation ability, adult stem cells have the advantages of being easier to obtain and having fewer associated ethical issues than embryonic stem cells. Recently, research on stem cells derived from oral tissues, including periodontal ligaments, has been reported. Although the interaction between ectodermal mesenchymal stem cells and epithelial stem cells is very important throughout the process of tooth development, there are difficulties in researching the interaction between the epithelium and mesenchyme for dental regeneration since epithelial stem cells are relatively difficult to obtain and maintain. Therefore, in this study, I established cell lines of Hertwig’s epithelial root sheath cells/epithelial rests of Malassez cells (HERS/ERM cells) and dental pulp epithelial stem cells (DPESCs), and investigated the effect of conditioned medium derived from these dental epithelial stem cell lines on the hard tissue forming ability of Stem cells from human exfoliated deciduous teeth (SHEDs). SHEDs and DPESCs were isolated and cultured from deciduous teeth, and HERS/ERM cells were isolated and cultured from permanent teeth. The obtained HERS/ERM cells and DPESCs were immortalized by introducing the SV40 large T antigen. In addition, a more stable cell line was established by introducing the human telomerase reverse transcriptase (hTERT) gene into the HERS/ERM cell line established with the SV40 large T antigen. To confirm whether the established cell line maintains its dental epithelial stem cell characteristics, FACS analysis of markers of embryonic stem cells and epithelial stem cells was performed to evaluate changes in cell morphology and gene expression when the epithelial-mesenchymal transition (EMT) was induced by TGF-β1. To confirm the interactions between dental epithelial stem cells and mesenchymal stem cells in hard tissue formation, the effects of conditioned medium obtained from primary dental epithelial cells and the cell lines on the hard tissue-forming ability of mesenchymal cells were examined. The immortalized dental epithelial cell lines had an extended life-span, and their morphology was maintained throughout subculture. RT-PCR data showed that the immortalized cell lines had typical epithelial stem cell-like gene expression patterns identical to those of primary dental epithelial cells. In addition, primary dental epithelial cells and the immortalized cell lines exhibited similar characteristics when the epithelial-mesenchymal transition was induced by TGF-β1. Alizarin red S staining indicated that calcium accumulation in SHED cells was equally promoted by conditioned medium derived from primary dental epithelial cells and the immortalized cell lines. Taken together, I established dental epithelial stem cell lines and confirmed that both the primary dental epithelial stem cells and the cell lines showed the same morphological characteristics and gene expression profiles. Furthermore, it was also proven that conditioned medium from the established cell lines affects the acceleration of the hard tissue formation of primary cultured mesenchymal stem cells, SHEDs. These data suggested that HERS-SV40, HERS-SV40 / hTERT and DPESC-SV40, which were established from primary dental epithelial cells, could be expected to contribute to the study of dental stem cell functions and tooth regeneration

A role of TGF-β1 in cancer epithelial-mesenchymal transition into mesenchymal-epithelial transition theory and molecular mechanism in lung cancer cells

김성관 건국대학교 대학원 2020 국내박사

상피간엽전환 (Epithelial-Mesenchymal Transition, EMT) 이론은 암의 전이를 설명하는 중요이론 중 하나이다. 주로 transforming growth factor-beta 1 (TGF-β1, TGF)을 통하여 EMT를 유도하는 것이 일반화되어 있다. 그러나 TGF는 다기능을 가진 신호전달물질로, 암세포의 microenvironment에서 어떤 영향을 줄 수 있을지 단정하기 어렵다. 또한 EMT의 역작용인 간엽상피전환 (mesenchymal-epithelial transition, MET)에서의 신호전달은 거의 알려져 있지 않다. 이 논문에서는 폐암세포주에 TGF를 연속적으로 투여하고, 제거하는 방법 이용하여 EMT, MET와 유사한 실험모델을 만들었다. 파트1에서는 A549 폐암세포주를 이용하여 EMT와 MET 모델을 만들고 그 특성을 확인하였다. 파트2에서는 네가지 종류의 폐암세포주 (A549, H23, H358, and H1299)를 이용하여 TGF의 공통된 작용이 무엇인지 확인하는데 중점을 두었다. 파트 1에서, 단백질 정량분석 실험결과에서, TGF를 처리한 그룹에서는 높은 Vimentin 발현과 낮은 E-cadherin 발현을 보였고, 반면 TGF를 제거한 그룹에서는 반대의 경향을 보였다. 암줄기세포의 표지물질의 변화에서는 TGF 처리한 그룹에서 CD24가 적게 발현하는 세포가 감소하였다. 더욱이 G2/M기의 세포들이 증가하고, cisplatin에 대한 민감도가 올라갔으며, 세포의 성장 및 이동능력이 향상되었다. 이러한 특징들은 TGF를 제거한 실험군에서는 다시 원래의 세포가 가지고 있던 능력대로 돌아가지는 않았다. 면역항체를 이용한 단백질 확인 실험에서 TGF를 처리한 실험군에서는 SMAD3, p-SMAD3, p-ERK, caspase-3가 증가하고, caspase-3 활성화가 감소하였다. 파트2에서, TGF를 처리한 그룹에서 A549, H23, H358 폐암세포주의 모양이 방추형으로 변형하였다. TGF를 제거한 그룹에서는 A549와 H23만이 원래 가지고 있던 형태로 다시 변형되었다. 이 결과는 E-cadherin과 Vimentin의 단백질 정량분석 결과로 뒷받침되는데, A549와 H23에서는 EMT와 MET 유사한 결과를 보였지만, H358은 EMT만 일어나는 듯한 결과를 보이고, H1299는 MET만 일어나는 결과를 보였다. 유세포분석결과에서는 A549와 H23에서 CD44의 레벨이 TGF를 처리했을 때 변화하였다가, TGF를 제거하였을 때 다시 원래대로 돌아가는 결과를 보였지만, H358과 H1299에서는 각각 CD24와 CD44의 변화가 TGF를 제거한 실험군에서 원래의 세포와 다른 결과를 보였다. 세포자연사 (Apoptosis)와 관련된 141개의 억제제를 이용한 기전 스크리닝 연구결과에서 Bcl-2, p53, TNF-α, MDM2, IAP, PD-1, Survivine, Nucleophosmin, MEK, GSK-3에 모든 실험군에서 연관이 되었고, MDM2, Survivine, Nuclophosmin, IAP, Bcl-2, MERTK 기전이 TGF-β1을 처리하였을 때 변화하였으며, Mcl-1, caspase3, EGFR, Nrf2, ERK, and MDM2 기전이 TGF-β1을 제거하였을 때 연관이 있었다. 전반적으로 이 결과들을 통해 TGF-β1이 항상 여러 종류의 폐암세포주에서 EMT나 MET를 유도하지는 않을 수 있으나, TGF-β1이 세포자연사 기전을 통해 폐암세포주에 세포 microenvironment인 면에서 역할을 할 수 있다는 가능성을 확인하였다. The Epithelial-Mesenchymal Transition (EMT) theory is one of the major mechanisms in cancer metastasis. Primarily, transforming growth factor-beta 1 (TGF-β1, TGF) has been used continuously as an inducer of EMT. TGF, however, is also a multi-functional cancer signaling molecule, which is difficult to precisely determine how it affects the multiple mechanisms present in the cancer cell microenvironment. Also, mesenchymal-epithelial transition (MET), which is the reverse mechanism of EMT, is scarcely known in TGF signaling. In this study, lung cancer cell lines were used to reproduce EMT and MET like model by TGF treatment and removal. The EMT and MET model was established using the A549 lung cancer cell line by TGF treatment and removal in part1. The role of TGF was investigated by using four types of lung cancer cell lines (A549, H23, H358, and H1299) in part2. Western blot analysis revealed that the TGF treated group showed high vimentin and low E-cadherin expression, whereas the TGF removed group showed the reverse trend in A549. Among cancer stem cell markers, the population of CD24low cell was reduced in the TGF treated group of A549. Furthermore, the G2/M phase cell cycle population, cisplatin-sensitivity, and cell proliferation and migration ability were increased in the same group. These features were unaltered in the TGF removed group in A549 when compared to the TGF treated group. Immunoblotting showed an increase in the levels of SMAD3, phosphorylated SMAD3, phosphorylated ERK, and caspase-3, and decrease in the active caspase-3 levels in the TGF treated group of A549. In part2, Cell shapes of A549, H23, and H358 were changed to spindle-like forms in the TGF treated group. In the TGF removed group, the A549 and H23 cell line returned to its original round-like cell shape. These results were supported by western blot assay of E-cadherin and Vimentin in which A549 and H23 showed EMT and MET like properties. However, H358 showed only EMT like properties, and H1299 solely showed MET like properties. In flow cytometry results, A594 and H23 showed changes in CD44 levels after TGF treatment and returned to its original cell line profile after TGF removal. However, H358 and H1299 showed only small changes in CD24 and CD 44 levels respectively, after TGF removal without returning to its original profile. The results from 141 apoptosis-related inhibitor assays showed that Bcl-2, p53, TNF-α, MDM2, IAP, PD-1, Survivine, Nucleophosmin, MEK, and GSK-3 signalings were initially being associated with the four cell lines used in this study, whereas MDM2, Survivine, Nuclophosmin, IAP, Bcl-2, and MERTK mechanisms were altered by TGF-β1, and Mcl-1, caspase3, EGFR, Nrf2, ERK, and MDM2 signalings were influenced by TGF-β1 elimination. Overall, the results indicated that although TGF-β1 could not fully lead EMT or MET in various types of lung cancer cells, it could still play a prevalent role in apoptosis mechanisms in the cancer cell microenvironment.

Human Dental Epithelial Cells Induce Odontogenic Differentiation of SHED

채근영 서울대학교 대학원 2019 국내석사

Tooth organogenesis and regeneration occur through reciprocal interaction between epithelial and ectomesenchymal stem cells. This cell-to-cell communication is also a key regulator in the differentiation of ameloblasts and odontoblasts which secrete enamel and dentin, respectively. In studying tooth regeneration, the dental pulp has been widely used as the source of mesenchymal stem cells. On the contrary, most of the epithelium is lost after tooth eruption and root completion, and thus have not been vastly explored. Recently, prior research has characterized dental epithelial cells known as Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM) extracted from human periodontium and its cell line was established. However, the epithelial-mesenchymal signaling capacity of HERS/ERM has yet to be elucidated. Thus, this study was conducted to elucidate the effect of HERS/ERM conditioned medium (CM) on the odontogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED). To simulate the effect of epithelial paracrine actions on mesenchyme, SHED were cultured in differentiation medium supplemented with varying proportions of CM derived from the HERS/ERM cell line. The CM collected was freeze-dried for further concentration of the solution, and the potency of CM was tested at 1X (differentiation medium supplemented with 10 v/v% CM), 4X and 8X concentration factors, using freeze-dried CM for the 4X and 8X treatments. To determine the effects of the varying concentration of CM, Alizarin red S staining, RT-qPCR, Western blot, and DAPI nuclear staining were assessed and the results were compared to SHED treated with the freeze-dried basal media of CM. Alizarin red S staining revealed that increasing the concentration of CM treatment had a distinctive impact on the amount of calcium nodule formed in the differentiated SHED. Expression levels of mineralization-related markers, RUNX2, BSP, DMP1, ON, OC, and MEPE also confirmed the enhanced odontogenic effect of CM concentration on day 8 and 12 as CM concentration was increased. In contrast, the addition of freeze-dried basal media exhibited a lack of calcium nodule formation and no significant changes in the mineralization-related mRNA expression levels. The expression of dentin phosphoprotein, an odontoblast marker observed by Western blot, was also more prominent in CM-treated SHED than in basal media-treated SHED on both day 16 and 20. Also, long-term culture of SHED with CM exhibited cell death, whereas the basal media control group did not. The DAPI nuclear staining revealed a phenomenon similar to the terminal differentiation of cells. The data indicate that CM from human HERS/ERM cell line has odontogenic differentiation capabilities that are concentration-dependent, and further investigations may contribute to the discovery of specific growth factors and cytokines at play. This study is the first report of odontogenic induction potential of concentrated CM derived from the human dental epithelial cell line. 치아 발생과 재생 과정은 상피와 외배엽성 간엽 줄기세포 간의 다양한 상호작용으로 이루어지며 치아를 구성하는 법랑질 및 상아질은 이러한 상피-간엽 상호작용을 통해 분화된 법랑모세포와 상아모세포로부터 형성된다. 현재까지의 치아재생 연구는 분리 및 유지가 용이한 치수 간엽줄기세포를 중심으로 이루어졌는데, 반면에, 치아 상피세포는 치아 발생이 끝나고 나면 대부분 소실되기 때문에 확보 및 활용이 어려워 관련 연구가 많이 이루어지지 못하였다. 최근 선행 연구를 통해 사람 치주조직으로부터 치아 상피세포인 Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM)가 확보되었으며 HERS/ERM 세포주 또한 확립되었으나 아직까지 치아조직재생에 대한 HERS/ERM의 역할에 대해서는 충분한 검증이 이루어지지 않았다. 본 연구에서는 사람 유치 치수줄기세포(stem cells from exfoliated deciduous teeth, SHED)에 HERS/ERM 세포주의 조건배지를 처리한 후 SHED의 상아모세포 분화 능력을 관찰함으로서 치아재생 과정의 상피-간엽 상호작용에서 HERS/ERM의 역할을 확인하고자 하였다. 사람 유치 치수에서 SHED를 일차 배양하였고, HERS/ERM 세포주를 48시간 동안 배양하여 얻은 조건배지를 SHED에 10, 40, 80%(v/v)로 처리하였다. 다만, 조건배지를 40%와 80%로 추가할 때에는 용량을 최소화시키기 위해 조건배지를 동결건조로 농축시킨 후 사용하였다. 조건배지를 처리한 SHED는 Alizarin red S staining, RT-qPCR, Western blot 및 DAPI 핵염색을 시행하여 상아모세포로의 분화 유무를 확인하였고, 세포배양 과정을 거치지 않은 조건배지의 basal media를 추가하여 대조군으로 사용하였다. Alizarin red S 염색 결과 조건배지의 농도가 증가함에 따라 12일에서 20일까지 칼슘 침착이 증가하는 것을 볼 수 있었다. 경조직의 특이 유전자인 BSP, DMP1과 MEPE의 발현도 조건배지의 농도가 증가함에 따라 8, 12일에 상승하였고, RUNX2, ON과 OC는 줄어드는 양상을 보였다. 반면에, 대조군으로서 조건배지의 basal media만 추가한 경우 Alizarin red S 염색이 되지 않았으며 유전자 발현 양상도 분화에 따른 변화가 없었다. 상아모세포 표지단백질인 dentin phosphoprotein 단백질 또한 16, 20일간 조건배지가 처리된 SHED에서 발현됨을 확인하였고, 대조군에서보다 더 많이 발현되는 것을 확인하였다. SHED에 조건배지를 처리하면서 장기간 배양하였을 때 대조군과는 달리 세포가 죽는 현상이 관찰되었고, DAPI 핵염색을 통해 관찰하였을 때 terminal differentiation과 연관있는 cell death와 같은 현상임이 추정된다. 이상의 연구결과를 종합하면 사람 HERS/ERM 세포주의 조건배지가 SHED의 상아모세포 분화를 유도하며 조건배지의 농도가 증가함에 따라 분화시기를 앞당기고 분화효율이 향상됨을 알 수 있었다. 따라서 HERS/ERM이 SHED의 상아모세포 분화에 미치는 영향은 조건배지를 통하여 상피에서 간엽간으로 정보가 전달됨으로써 이루어지는 것을 확인하였으나 분화유도물질의 발굴 및 동정과 같은 후속연구가 필요할 것이다.

The role of TGF β1 on epithelial-mesenchymal like transition in cancer cells

이재연 이화여자대학교 대학원 2000 국내박사

TGFβ1은 multifunctional cytokine으로서 발생 과정과 상처 치유, 면역 반응, 암세포의 침윤 과정 등에서 여러 기능을 수행하는 것으로 알려져 있다. 본 실험에서는 자궁경부암에서 유래한 SiHa 세포주를 사용하여 epithelial-mesenchymal 전환의 측면에서 TGFβ1의 영향을 조사하였다. 3차원 배양에서 TGFβ1을 농도별로 (0.1, 1, 10 ng/ml) 처리하였을 경우, SiHa 세포가 type I collagen gel matrix 안으로 농도 의존적으로 침윤되는 것이 관찰되었다. 2차원 배양에서도 3차원 배양에서와 같이 TGFβ1을 처리한 세포의 모양이 평평한 섬유아세포의 형태로 변화되는 것을 볼 수 있었다. TGFβ1은 세포간 물질의 발현에도 영향을 미치는 것으로 보여졌는데, fibronectin (FN)과 type I collagen (Col I)의 발현은 증가시켰고, laminin (LN)과 vitronectin (VN)의 발현은 별다른 영향을 받지 않았다. FN의 matrix assembly 또한 TGFβ1에 의해 유도되어 졌다. α1, α6와 αv등의 integrin은 TGFβ1 처리로 세포 표면의 발현이 증가된 반면, FN 수용체인 α5β1은 세포간의 접촉 부위에서 세포와 기질간의 부착 부위로 재배열되는 것을 관찰할 수 있었다. Actin의 발현 정도는 TGFβ1의 처리와는 무관하게 보여졌으나, 그 분포는 변화되는 것으로 보였다. Phalloidin을 사용하여 actin filament를 염색한 결과, 세포간의 경계면에 존재하던 actin이 TGFβ1에 의해 stress fiber projection의 형태로 위치가 변화되는 것을 확인하였다. Focal adhesion을 구성하는 vinculin과 talin이 focal contact 부위로 재배열되는 것이 관찰되었으며, focal adhesion kinase (FAK)의 인산화 또한 증가되었다. 이러한 세포의 모양 변화가 epithelial-mesenchymal 전환과 관련이 있는지를 알아보기 위해 섬유아세포의 표지로 알려진 vimentin의 발현 유무를 조사하였으나, 발현되지 않는 것으로 확인되었다. 그러나, E-cadherin을 조사한 결과, epithelial-mesenchymal 전환 과정에서와 마찬가지로 TGFβ1 처리로 인해 발현이 감소되는 것으로 관찰되었다. TGFβ1에 의한 epithelial-mesenchymal 전환의 유도는 TGFβ1를 제거한 후에도 actin 재배열과 FN matrix 형성이 보여지기 때문에 비가역적인 현상으로 사료되며, 이는 3차원 배양에서 TGFβ1를 제거한 후 침윤현상이 지속되는 것으로도 뒷받침되었다. SiHa 세포의 epithelial-mesenchymal 전환 과정을 시간별로 관찰해 보면, actin 재배열은 6시간 처리에서부터 보여지지만 FN matrix 형성은 16시간 처리에서 관찰되므로 actin의 재배열이 FN matrix의 형성보다 선행하는 것으로 사료되었다. Actin의 재배열과 FN matrix의 형성이 상호 의존적으로 발생하는지의 여부도 조사 되였다. Actin filament의 형성을 저해하는 물질인 cytochalasin D의 전처리로 actin filament가 완전히 붕괴된 상태에서도 FN matrix가 TGFβ1에 의해 유도되었으며 recombinant human FN을 배양액에 첨가하였을 때에도 actin filament의 재배열은 일어나지 않았기 때문에 두 가지의 사건이 독립적으로 일어날 수 있다는 가능성을 제시하였다. 또한, recombinant human FN을 3차원 배양에서 배양액에 첨가하였을 경우, SiHa 세포의 침윤을 유도하는 것으로 관찰되었다. 정상 상태의 구강 점막 (OM) 세포와 형질 전환된 표피세포인 HaCat, 그리고 편평 상피암 세포주인 SCC13 등도 조사하였는데 SiHa 세포에서 관찰된 형태의 변화는 유도되지 않았다. Actin filament의 재배열과 integrin의 발현 위치 변화, FN matrix의 형성과 E-cadherin 발현 감소 등으로 대표되는 이러한 결과들은 TGFβ1에 의한 SiHa 세포의 모양 변화가 배 발생 단계에서 관찰되는 epithelial-mesenchymal 전환과 유사하다는 사실을 뒷받침한다. TGFβ1의 이러한 영향은 SiHa 세포에서 침윤 현상을 유도하는데 관여할 것으로 사료된다. TGF 1 is a multifunctional growth factor implicated in various physiological processes during development, wound repair, inflammation, immune system function and tumor invasion. In this study, we have investigated whether TGF 1 stimulates epithelial-mesenchymal transition (EMT) in SiHa cell line, derived from human cervical squamous carcinoma, which may positively affect invasive transition of tumors. In the raft culture, SiHa cells did not invade into type I collagen gel matrix but TGF 1 supplement (0.1, 1, 10ng/ml) stimulated the invasion in a dose-dependent manner. In submerged culture as well as raft culture, TGF 1-treated cells showed distinct morphological change, flattened fibroblastic type. TGF 1 stimulated expression of fibronectin (FN) and type I collagen (Col I) but not of other extracellular matrix (ECM) components, such as laminin (LN) and vitronectin (VN). Matrix assembly of FN was also observed in the TGF 1-treated SiHa cells. Cell surface expressions of integrin 1, 6 and v were increased based on surface biotinylation and immunoprecipitation analysis while FN receptor ( 5 1) was exclusively relocalized from cell-cell contact sites to cell-substratum attachment sites. Although SiHa cells showed similar levels of actin with or without TGF 1 treatment based on immunoblotting analysis, its spatial distribution within the cells was dramatically changed. Phalloidin staining showed that TGF 1 stimulated reorganization of actin filament from apical ring like structure to stress fiber projection. Vinculin and talin were relocated to focal contacts by TGF 1. Phosphorylation of focal adhesion kinase (FAK) was also increased upon TGF 1 treatment. To investigate whether these fibroblastic morphological changes are related to EMT, we examined the expression of vimentin which is a major intermediate filament of fibroblasts. Although vimentin was not detected at all, E-cadherin, one of the molecules that is down-regulated during EMT, was decreased. These effects of TGF 1 on epithelial-mesenchymal (EM) like transition appeared irreversible, since removal of TGF 1 after 5 min treatment did not stop the actin rearrangement and the FN matrix assembly. It was also confirmed in SiHa cell raft culture because invasion was induced by the transient exposure of TGF 1. In the time course of EM like change by TGF 1 treatment in SiHa cell line, actin rearrangement begin to appear at 6hr when FN matrix assembly has not been started yet. Therefore, actin rearrangement seems to precede FN matrix assembly. Furthermore, we investigated whether these two events are dependent each other or not. SiHa cells were pretreated with cytochalasin D before the addition of TGF 1. Cytochalasin D-treatment did not disrupt TGF 1-induced FN matrix assembly, even though actin filaments were completely disrupted at this condition. In the same context, supplementation of soluble recombinant human FN to the culture media did not cause rearrangement of actin filaments in submerged culture, and it was enough to induce invasiveness in raft culture. We also investigated other epithelial cells, normal oral mucosal (OM), HaCat and SCC13 cells, but could not observe the morphological changes as shown in SiHa cells. In conclusion, TGF 1-induced fibroblastic morphological change of SiHa cell line, which were confirmed by the relocalization of actin and integrins, the assembly of FN matrix and the down regulation of E-cadherin, is similar to EMT observed in the embryonic development. These effects of TGF 1 may play a positive role in invasive transition of SiHa cell line.

한쪽 요관 결찰한 신장에서 신장 근위상피세포 SIRT1 결핍의 신손상과 내형질 스트레스에 대한 효과

Tung, Nguyen Thanh 전북대학교 일반대학원 2017 국내박사

Renal fibrosis characterized by process of scar formation, is the final common pathway of chronic kidney diseases. The rodent model of unilateral ureteral obstruction (UUO) is considered a model of progressive renal fibrosis and inflammation, as well as a model of progressive renal fibrosis and inflammation. The proximal tubule epithelial cells are the most populous cell in the kidney. Recently, it has been reported that injury of proximal tubular epithelial cells alone triggers interstitial fibrosis. Therefore, targeted gene suppression of proximal tubular epithelial cells injury may attenuate progression of renal fibrosis. SIRT1 is a mammalian nicotinamide adenine dinucleotide-dependent protein deacetylase, and nicotinamide phosphoribosyltransferase, classified as class III histone deacetylases. SIRT1 has been demonstrated having a renal protective effects in various models of renal diseases. However, the role of renal tubular epithelial cells SIRT1on kidney fibrosis has not been elucidated yet. Hence, the present study was attempted to investigate the effect of proximal tubular SIRT1 on kidney injury and endoplasmic reticulum stress during UUO-induced renal fibrosis. We generated mice with conditional knockout of SIRT1 in renal proximal tubular epithelial cells by combining floxed Sirt1 and Cre recombinase under the control of gamma-glutamyl transferase promoter. The selective deletion of SIRT1 in kidney proximal tubular epithelial cells was confirmed with laser dissection and polymerase chain reaction. SIRT1 deletion in renal proximal tubule increases UUO-induced tubular injury score and depositions of type I collagen and fibronectin. Decrease of SIRT1 in proximal tubular epithelial cells enhanced UUO-induced epithelial–mesenchymal transition by increased α-SMA, increased vimentin and decreased E-cadherin expressions. Intercellular adhesion molecule-1 and monocyte chemoattractant protein-1, inflammatory response markers, significantly increased in conditional knockout UUO kidney, compared to those in control mice. Disruption of SIRT1 in the proximal tubular epithelial cells also enhanced renal tubular interstitium macrophage infiltration after UUO. Furthermore, knockout of SIRT1 in proximal tubulars epithelial cells showed increased expression of C/EBP homologous protein (CHOP), an endoplasmic reticulum stress response marker, and decreased expression of heme oxygenase-1 (HO-1) an antioxidant endoplasmic reticulum stress protein in UUO-operated kidney compared to sham-operated kidney. Thus, these finding indicate that SIRT1 deficiency in the proximal tubular epithelial cells promotes UUO-induced fibrosis with down-regulation of antioxidant endoplasmic reticulum stress.

TGF-β1-induces epithelial to mesenchymal transition (EMT) via ROS upregulation in human bronchial epithelial cells

김지은 건국대학교 대학원 2021 국내석사

형질전환성장인자-베타1(Transforming Growth Factor – beta1, TGF-β1)는 다능성 사이토카인으로, TGF-β 타입 1, 타입 2 수용체와의 결합 후 활성화되어 세포의 분화와 증식, 세포자멸사 등을 유도한다. 이가 인간 상피 조직에서 활성산소종(ROS)을 발생시키고, 상피-중간엽 전이(EMT)를 유도하여 조직 리모델링에 기여함이 최근 밝혀졌다. 이 연구에서 우리는 부비동의 염증과 상피조직 리모델링에 의한 비용종의 형성이 주요 특징인 비용종을 동반한 만성 비부비동염(CRSwNP) 환자에게서 TGF-β1과 그 수용체의 발현이 높은 것을 확인하여, 인간 기관지 상피세포인 BEAS2B 세포와 primary human nasal epithelial(NHNE)세포에 TGF-β1을 처리한 결과, ROS 발생과 그에 따른 세포의 세포자멸사가 증가하였음을 확인하였다. 추가적인 세포주기 확인 결과, TGF-β1에 의한 ROS는 G2/M 주기의 정체를 유도하였다. TGF-β1 처리 후 EMT 마커의 발현을 확인한 결과, 중간엽 마커의 발현은 증가하였고 상피 마커의 발현은 감소하였는데 이는 항산화제인 N-acetyl cysteine(NAC)와 함께 처리하였을 때 대조군과 비슷한 수준으로 회복하였다. 이러한 결과는 TGF-β1이 인간 기관지 상피에서 ROS 발생을 통해 세포 자멸사를 증가시키고 EMT를 유도하여 조직 리모델링을 통해 병증을 유발시킬 수 있음을 의미한다. Transforming Growth Factor – beta1 (TGF-β1) is a multifunctional cytokine that binds to TGF-β receptor types 1 and 2 and then activates to induce cell differentiation, proliferation and apoptosis. Recently it is being reported that TGF-β1 induces Reactive oxygen species (ROS) and Epithelial-mesenchymal transition (EMT) in human epithelial tissues, contributing to tissue remodeling. In this study, we identified high expression of TGF-β1 and its receptors in Chronic Rhinosinusitis with Nasal Polyps (CRSwNP) patients, where sinus inflammation and nasal polyp formation by epithelial tissue remodeling are the main features. Treatment of TGF-β1 on human bronchial epithelial cells, BEAS2B cells and primary human nasal epithelial(NHNE) cells, resulted in increased ROS production and subsequent apoptosis. In addition, as a result of cell cycle analysis, ROS by TGF-β1 induced G2/M arrest. As a result of confirming the expression of the EMT marker after TGF- β1 treatment, the expression of the mesenchymal marker increased and the expression of the epithelial marker decreased, which recovered to a level similar to that of the control group when treated with antioxidant N-acetylycysteine (NAC). These results suggest that TGF-β1 can Increase cell apoptosis via ROS development in human bronchial epithelium and induce EMT to induce pathology through tissue remodeling.

Molecular Modulation and Differential Characterization of Mammary Adipose Stem Cells towards Epithelialization

Reza, Abu Musa Md. Talimur Kangwon National University 2014 국내박사

The research was designed to investigate the changes in gene expression pattern of mammary gland epithelial cells and adipose stromal cells with the changes in cellular microenvironment through epithelial induction mixture, adipogenic mixture and angiogenic mixture and also to justify the potential use of mammary adipose-derived stem cells for the regeneration of mammary glandular epithelial-like cells. Therefore, I conducted the whole studies under three different chapters. Chapter-I says about “Differentiation of adipose cells to epithelial lineage and epithelial cells to adipogenic lineage”. Chapter-I was composed of three different independent experiments, where the first experiment was conducted using mammary fat pad adipose derived stem cells isolated from Korean Black Goat mammary fat pad adipose tissues. The second and third experiment was conducted using 3T3L1 mouse adipose cells and MAC-T bovine mammary epithelial cells respectively to support our findings from the first experiment. The Chapter-II says about “Modulation of gene expression in mammary epithelial cells using angiogenic factors in presence or absence of mammary adipose stromal cells”. The chapter-II was composed of a detailed experiment to investigate the effect of angiogenic mixture for the differentiation of mammary epithelial cells in presence or absence of mammary adipose stromal cells. Finally, the Chapter-III says about “Transfection of K18 gene to CD44+ rat mammary adipose derived cells and subsequent infusion of the transfected cells to the rat aural space for epithelializaion”. Chapter-III basically investigated the transfection potential of mammary epithelial-specific gene(s) to mammary adipose-derived stem cells for the generation of mammary epithelial-like cells. In Chapter-I, the Experiment-I was conducted to know and investigate the mechanism involved during mesenchymal to epithelial transition to unravel questions related to mammary gland development in prepubertal Korean black goat. We therefore, biopsied mammary fat pad and isolated adipose cells and characterized with stemness factors (CD34, CD13, CD44, CD106 and vimentin) immunologically and through their genetic expression. Furthermore, characterized cells were differentiated to adipogenic (thiazolidenediones and α-Linolenic acid) and epithelial (keratinocyte growth factor) lineages. Thiazolidenediones /or in combination with α-Linolenic acid demonstrated significant up-regulation of adipo-Q, PPAR-γ, CEBP-α, LPL, and resistin. Adipose stem cells in induction mixture (5 μg/ml Insulin, 1 μg/ml Hydrocortisone, 10 ng/ml epidermal growth factor) and subsequent treatment with 10 ng/ml keratinocyte growth factor revealed their trans-differentiating ability to epithelial lineage. From 2 d onwards, the cells under keratinocyte growth factor influenced cells to assume rectangular (2-4 d) to cuboidal (8-10 d) shapes. Ayoub-shklar stain developed brownish-red pigment in the transformed cells. Though, expressions of K8 and K18 were noted to be highly significant (p<0.01) but expressions of epithelial membrane antigens (EMA) and epithelial specific antigens (ESA) were also significant (p<0.05) compared to 0 d. Conclusively, epithelial transformations of mammary adipose stem cells would add up knowledge to develop therapeutic regimen to deal with mammary tissue injury and diseases. Experiment-II was conducted for the further justification of the findings of Experiment-I. Aiming at mesenchymal to epithelial transition, we investigated the potential of keratinocyte growth factor (KGF) and bone morphogenetic protein (BMP)-6 separately for the induction of mesenchymal to epithelial transition in 3T3L1 mouse adipose cells and to trace the molecular events that probably upregulates during mesenchymal to epithelial transition induction. KGF successfully induced mesenchymal to epithelial transition through the up regulation of epithelial related genes and transcript expression on 3T3L1 cells. In contrast, BMP-6 plays completely reverse role through down regulation of all epithelial related genes and transcript expression. In KGF based treatment, seven genes (K8, K18, EpCAM, K5, K14, SMN1 and α-SMA) out of total eight genes were significantly (p<0.05/ p<0.01) up regulated. Immunostaining and immunoblotting also revealed significant (p<0.05/ p<0.01) expression of several epithelial-specific surface antigens and transcripts. Moreover, ayoub shaklar staining (specific to keratin) of KGF treated cells showed formation of keratin (reddish brown color) within cytoplasm of the cells whereas, the control and BMP-6 treated cells did not. Conclusively, KGF was observed to have the potential to enhance mesenchymal to epithelial transition and these clues could be used in the future research of cellular reprogramming and regenerative medicine Experiment-III was performed to investigate the effect of adipogenic mixture for the differentiation and formation of cytoplasmic lipid droplets (CLD’s) in MAC-T mammary epithelial cells. CLD’s formation is a critical requirement for the onset of lactation and prevention of many diseases in the mammary gland. We therefore investigated the number of genes and transcripts that could be detrimental in upholding the formation of CLD’s in bovine mammary epithelial cells (MAC-T). We therefore, designed the present research to investigate the effect of alpha-linolenic acid (LA) plus Thiazolidenediones (TZD’s) in the formation of CLD’s in bovine mammary epithelial cells (MAC-T). MAC-T cells were proliferated in defined medium and upon confluence treated with differentiation medium containing LA (100 μM) and TZD’s (10 μM), and the medium was changed at every 48 h until 8 d. Control cells were cultured only in DMEM+2 % HS+1.1 % PS. Microscopically, it was quite apparent that LA plus TZD’s had successfully introduced CLD’s in cellular cytoplasm and that was further confirmed with Oil-red-O staining that showed significant (p<0.05) elution index signifying the amount of stain retained within. We also observed significant (p<0.05/P<0.01) up-regulation of adipogenic and down regulation of epithelial markers. In conclusion, 100 μM LA plus 10 μM TZD’s results formation of CLD’s and subsequent differentiation of MAC-T cells. In addition, evidence has been presented here that TZD’s and LA could absolutely alter and initiates a cellular spark in the mammary epithielial cells to undergo concerted with adipose program and develop shifts in the lipid metabolism to develop CLD’s efficiently and to increase the availability of fatty acids necessary for lipid synthesis by milk secreting cells. In Chapter-II, the investigation was performed to find out the effect of angiogenic factors for the differentiation of MAC-T mammary epithelial cells in presence or absence of mammary adipose stromal cells under different culture models. Successful lactation mainly influenced by three major factors such as growth, proliferation and differentiation of mammary epithelia, proper functioning of stromal cells and abundant blood circulation. Thus, the balance in the presence of epithelial cells, adipose cells, endothelial cells, nerve cells and immune cells and their synergistic influence results a successful lactation. We believed that further understanding the influence of angiogenic factors in mammary parenchyma-stromal cells interaction would explore the hidden regulatory mechanism for the proliferation and differentiation of mammary epithelial cells. Therefore, we also designed our study to determine the effect of angiogenic factors VEGF plus T4 and/or Tetrac (angiogenic blocker) in MAC-T bovine mammary epithelial cells in three different culture conditions such as monolayer culture of MAC-T cells, trans-well culture (adipose at top and MAC-T at bottom) and mixed-culture (1:1 mixture of adipose and MAC-T cells). Both epithelial and adipose cells were seeded with a density of 2×106 cells per well in DMEM+10% FBS+1.1% PS and incubated for 24 h. Thereafter, the cells were exposed to angiogenic induction medium (DMEM+2%HS+1.1% PS+10 ng/mL bFGF+10 ng/mL EGF) for another 48 h. Finally, the cells were cultured again 48 h with four different treatment combinations (such as 50 ng/ml VEGF, 50 ng/ml VEGF+100 nmol/L T4, 50 ng/ml VEGF+10-7 M Tetrac and 50 ng/ml VEGF+100 nmol/L T4+10-7 M Tetrac) and one untreated control. The treated cells were harvested subsequently and were subjected to different analytical methods. In monolayer culture, only angiogenic markers and some epithelial markers showed significant up-regulation (P<0.05/P<0.01), and mammary epithelial-specific Keratin-18 (K18) got enhanced insignificantly with VEGF+T4 treatment and seemingly, 10-7 M tetrac is not enough to block the effect of angiogenic factors. In trans-well culture, only VEGF treated cells significantly (P<0.05/P<0.01) up-regulated few angiogenic transcript and down-regulated few epithelilal markers. VEGF+ T4 treated cells showed significant (P<0.05/P<0.01) up-regulation of most of the angiogenic and adipogenic markers and but down-regulated the mammary epithelial-specific marker K18. In this group also 10-7 M tetrac could not block the angiogenic effect but reduced the effect of angiogenic factors in different extent. In mixed-culture cells, most of the genes and markers lose their expression however, in control cells there was weak expression for different markers and epithelial marker K18 totally disappeared. However, VEGF+ T4 treated cells showed significant (P<0.05/P<0.01) up-regulation for few angiogenic and epithelial and in some cases adipogenic markers under different analytical methods. The cells treated in combination with blocker (Tetrac) caused inconsistent up/down-regulation of some transcript compared to the control cells, but it appears that the homogenous and direct haphazard mixture resulted the loss of characteristics features of mammary epithelial cells. Furthermore, the microarray gene expression profile was checked for the co-cultured mammary epithelial cells. The microarray gene profiling showed, the VEGF alone mainly influenced the expression for adipogenic differentiation genes, lipid metabolism gene expression, and also cause reduction in the expression of cell adhesion molecules. VEGF+T4 treatment resulted up-regulation of endothelial cell adhesion molecules, cell proliferation molecules, and down-regulation of lipid metabolism molecules. Furthermore, VEGF alone up-regulated PPAR signal (specific for adipogenesis) and VEGF+T4 up-regulated WNT signal (known for endothelial growth and proliferation). Therefore, it could be concluded that VEGF alone might pose the role towards differentiation of the mammary epithelial cells and VEGF+T4 could influence the cells transformation towards endothelial-like cells. The angiogenic factors could not cause the loss of epithelial markers in absence of adipose cells. But, the systematic presence of adipose factors could influence the characteristics features of mammary epithelial cells and the haphazard and direct mixture of mammary epithelial cells with adipose stroma could cause the loss characteristics markers expression. In Chapter-III, the research was conducted focusing on transfection of mammary epithelial-specific gene to mammary adipose stroma derived stem cells for the investigation of the potential uses of mammary adipose-derived stem cells for the regeneration of mammary epithelial cells. In this study, the mammary adipose tissue were surgically harvested from Slc:SD female rat of 10 week old (around 200 g live weight) and the adipose-derived cells were isolated using enzymatic digestion process with collagenase type-I and then comparatively smaller sized cells were collected through differential trypsinization process. After that, the mesenchymal stemness marker CD44+ cells were sorted using FACSAria-II. Finally, the CD44+ cells were transfected with mammary epithelial specific gene K18 and subsequently the transfection was confirmed by detecting GFP+ cells through fluorescence microscopy and thereafter, the downstream analysis of the K18 gene transfected cells was performed by puromycin antibiotic treatment, gene and protein expression profiling, Ayoub shklar staining and finally the cells were grafted to the rat aural space for in vivo study. In result, 25-30 % cells were found to be transfected when it was detected for GFP+ expression under fluorescence microscopy after 24 h of transfection. But, the gene and protein expression did not show significant difference between control and transfected cells. However, the ayoubl shklar staining showed reddish brown color formation in some cells, but all of the cells had shrinkage cytoplasm (spider shape) and seemed to get detached chronologically from the surface of the culture dish. Therefore, it could be concluded that the conversion of mammary adipose CD44+ cells towards epithelial lineage need to be standardized further for having sound transfection rate and subsequent survivability of the cells.

Development and Characterization of Dental Epithelial Cell Lines from Human Induced Pluripotent Stem Cells

기민지 서울대학교 대학원 2022 국내석사

치아의 발달 및 재생 과정에서 치아 상피세포와 외배엽성 중간엽세포의 상호작용은 중요한 요소이기 때문에, 치아재생연구에서 치계상피세포와 중간엽세포의 확보는 필수적이다. 하지만, 다양한 세포원으로부터 비교적 확보가 쉬운 중간엽 줄기세포와는 달리, 사람 치아 상피줄기세포는 치아가 맹출되면 소실되는 특성 때문에 확보 및 배양이 어렵다. 현재까지 보고된 치아 상피줄기세포는 영구치 치주 조직으로부터 분리된 Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM cells) 과 유치 치수로부터 분리된 Dental pulp epithelial stem cells (DPESCs)가 있으나 두 종류의 세포 모두 수명이 짧기 때문에 안정적인 유지에 어려움이 있다. 최근 선행 연구를 통해 HERS/ERM cell을 지지세포층으로 사용하여 사람 유치 치수줄기세포(dDPSC) 유래 유도만능줄기세포를 분화시킨 치아 상피 유사 세포주가 보고되었다. 본 연구에서는 이 선행 연구의 방법을 활용하여 다양한 기원의 치아 상피줄기세포를 확보하고 특성을 검증하고자 하였다. 이를 위해 확보가 비교적 용이하고 유도만능줄기세포 확립 연구가 많이 이루어진 혈액 유래 PBMCs에서 확립한 유도만능줄기세포를 치아상피세포로 분화시키기 위해 HERS/ERM cells 지지세포 위에 배양하였다. 또한 지지세포를 다양화하기 위해 dDPSC 유래 유도만능줄기세포를 DPESCs 지지세포 위에 배양하여 치아상피세포로 분화 유도하였다. 서로 다른 기원의 유도만능줄기세포와 지지세포를 사용하여 확립된 두 상피줄기세포주의 특성을 확인하였으며, 새로운 지지세포위에서 분화된 상피세포주는 분화능을 확인하기 위해 dDPSCs와의 공배양을 통하여 치아모세포로 분화 유도하였다. PBMCs 및 dDPSCs 유래 유도만능줄기세포는 14일 동안 각각 HERS/ERM cells 및 DPESCs 지지세포층 위에서 배양하여 분화 유도하였고, 분화된 세포는 SV40 Large T antigen을 도입하여 불멸화하였다. 확립된 PBMC-iPSC-derived epithelial-like cells on HERS/ERM (PHes)와 dDPSC-iPSC-derived epithelial-like cells on DPESC (DDes)는 장기배양 후에도 세포 형태의 변화가 없이 일정하게 증식이 유지되었다. 또한, STR 분석으로 확립된 세포주의 기원이 각각의 iPSC임을 검증하였으며, flow-cytometry를 통한 세포표면항원분석, PCR을 이용한 상피줄기세포 및 배아줄기세포 유전자 발현 분석으로 상피세포의 특성을 지님을 확인하였다. DDes는 dDPSCs와 공배양하며 8일간 경조직 분화를 유도하였을 때 법랑모세포 관련 유전자인 Enamelin, KLK4와 상아모세포 관련 유전자인 Runx2, BSP, ONN, OCN, DMP1, MEPE, Col1a1, DSPP 발현이 대조군에 비해 모두 증가하였으며 8일간 분화 후 DPP 및 enamelin 단백질 발현도 대조군에 비해 증가함을 확인하였다. 또한 분화 유도 20일 차부터 칼슘이 침착됨을 확인하였다. 이 결과를 통해, PBMC 유래 유도만능줄기세포와 dDPSC 유래 유도만능줄기세포를 각각 HERS/ERM cell과 DPESCs를 지지세포층 위에서 성공적으로 치아 상피줄기세포로 분화하였으며 새로운 지지세포층을 사용한 치아 상피줄기세포주의 치계세포 분화능도 검증되었다. 따라서 더 다양한 기원의 유도만능줄기세포와 지지세포층을 활용하여 분화능을 지닌 치아 상피줄기세포의 확립이 가능할 것이며 이는 부족한 치아 상피줄기세포 확보 문제를 극복할 수 있을 것으로 기대된다. Tooth development and regeneration are essentially regulated through epithelial-mesenchymal interactions between dental epithelium and mesenchyme. Due to the consequences of the development of teeth, dental mesenchymal stem cells have been obtained from various cell sources. However, it is difficult to isolate and maintain human dental epithelial cells because they were lost after the tooth eruption. Therefore, acquiring epithelial and mesenchymal stem cells for tooth regeneration is essential. The previous study has reported on the source of an epithelial stem cell by differentiating iPSCs: dental pulp stem cells from human exfoliated deciduous teeth (dDPSCs)-derived iPSCs differentiated into dental epithelial-like cells using Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells as a feeder. This study was conducted to develop alternative sources of dental epithelial stem cells for dental regeneration. In this study, differentiation of peripheral blood mononuclear cells (PBMCs)-derived iPSCs on HERS/ERM cells as a feeder and dDPSCs-derived iPSCs on DPESCs as a feeder was investigated. Each dental epithelial-like cell line was established and characterized. Dental differentiation was also examined by co-culture of the epithelial-like cells and dDPSCs. Differentiating PBMCs and dDPSCs-derived iPSCs into epithelial-like cells was induced on HERS/ERM cells and DPESCs feeder layers for 14 days, respectively. Differentiated epithelial-like cells were immortalized, and PBMCs-iPSCs derived epithelial-like cell lines (PHes) and dDPSCs-iPSCs derived epithelial-like cell lines (DDes) were established. STR analysis verified that each cell line originated from each iPSC, respectively. The immunophenotypes of PHes and DDes were similar by FACS analysis, and both had the characteristics of epithelial stem cells with expressions of epithelial stem cell- and stemness-related markers. Moreover, DDes co-cultured with dDPSC showed the potential for dental differentiation: the ameloblast-related genes and the odontoblast-related genes were highly expressed. The protein expression of the dentin phosphoprotein and enamelin intensely increased. In addition, mineralization in the co-culture group increased by odontogenic induction. Taken together, the established epithelial-like cells, DDes and PHes, had the characteristics of dental epithelial stem cells. Also, the co-culture of DDes and dDPSCs showed the capacity for dental differentiation. Therefore, various iPSCs-derived epithelial-like cell lines were established, and these cell lines are expected to be used in tooth regeneration.

Epithelial-Mesenchymal Transition-mediated regulation of gefitinib resistance and invasion through three dimensional (3D) collagen gels

이미숙 서울대학교 대학원 2014 국내박사

Normal epithelial cells are attached with one another or to the extracellular environment by cell-cell or cell-extracellular Matrix (ECM) interactions, respectively. By accumulated genetic alterations or other factors, the epithelial cells can become tumorigenic, with forming heterogeneous tumor masses. Among the tumor cell mass, certain metastatic cells lost their cell polarity and cell-cell adhesions, being converted to mesenchymal-like cells. This epithelial-mesenchymal transition (EMT) is critically involved in the cancer metastasis. This EMT process also causes resistance of cancer cells to anti-cancer reagents, leading to an attenuation of drug efficiency and enhances migration and invasion for metastatic potential of cancer cells. In this study, I investigated how EMT causes gefitinib resistance of NSCLC by virtues of TM4SF5 (transmembrane 4 L6 family member 5)-mediated EMT induction in NSCLC cells, and how EMT-rendered mesenchymal properties of cancer cells could regulate invasion of TM4SF5-positive and E-cadherin negative to be mesenchymal cells-like, through 3D collagen I gel systems. One of the most important pathways in NSCLC is the epidermal growth factor receptor (EGFR) pathway during tumor progression. In many cases, NSCLC patients can be initially treated with the EGFR-TKI (Tyrosine Kinase Inhibitor), gefitinib. However, continued gefitinib therapy does not benefit the survival of patients due to acquired resistance through additional EGFR mutations, c-MET amplification, or EMT. It is of further interest to determine whether mesenchymal-like, but not epithelial-like, cancer cells can become resistant to gefitinib by bypassing EGFR signaling and acquiring alternative routes of proliferative and survival signaling. Here I examined whether gefitinib resistance of cancer cells can be caused by TM4SF5, which has been shown to induce EMT via cytosolic p27Kip1 stabilization. Gefitinib resistant cells exhibited higher and/or sustained TM4SF5 expression, cytosolic p27Kip1 stabilization, and mesenchymal phenotypes, compared with gefitinib-sensitive cells. Conversion of gefitinib-sensitive to -resistant cells by introduction of the T790M EGFR mutation caused enhanced and sustained expression of TM4SF5, phosphorylation of p27Kip1 Ser10 (responsible for cytosolic location), loss of E-cadherin from cell-cell contacts, and gefitinib-resistant EGFR and survival signaling activities. Additionally, TM4SF5 overexpression lessened the sensitivity of NSCLC cells to gefitinib. Suppression of TM4SF5 or p27Kip1 in gefitinib-resistant cells via the T790M EGFR mutation or TM4SF5 expression rendered them gefitinib-sensitive, displaying more epithelial-like and less mesenchymal-like characteristics. These results indicate that TM4SF5-mediated EMT may have an important function in the gefitinib resistance of cancer cells. I have then investigated whether TM4SF5 induced EMT for enhanced cell migration and invasion to investigate how mesenchymal cell properties following EMT process may regulates the invasive properties such as invadopodia formation and ECM degradation in 3D collagen I-surrounded condition. The two dimensional (2D) cell culture systems can stratify neither the environments around in vivo cancer cells nor evaluation of efficacy of anti-cancer drug candidates with regards to cancer cell invasion and metastasis. For this reasons, I have used three dimensional (3D) cell culture systems using type 1 collagen matrices to study invasive behaviors and mechanisms of tumor cells. In this 3D cell culture system, revealing the TM4SF5-positive cell behaviors involved a technical problem of cell precipitation toward the bottom of the collagen I gels, leading to no-real 3D environment. Therefore, alternatively using highly-invasive and TM4SF5-positive MDA-MB-231 breast cancer cells, their invasive properties in 3D collagen I gels with normal serum-containing media were monitored for the underlying mechanisms. Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for our study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. I explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for snail1expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents. Altogether, this study reveals that mesenchymal properties acquired by EMT process, which can be induced by membrane proteins such as TM4SF5 can cause drug resistance and enhanced metastatic potentials of cancer cells, suggesting that EMT process can be targeted for anti-metastatic reagents.

Bortezomib Inhibits Proliferation, Migration, and Transforming Growth Factor-β1 Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells : 망막색소상피세포의 증식, 이동과 TGF-β1에 의해 유발된 상피-간엽 변형에 대한 Bortezomib의 억제효과

문건 계명대학교 대학원 2017 국내박사

Nuclear factor-kappa B (NF-κB) plays an important role in the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE). I investigated the effects of a proteasome inhibitor, bortezomib, on the EMT in RPE by regulating NF-κB expression. After treatment with various concentrations of bortezomib, cell population was analyzed by WST-8 assay, and cell-cycle regulation was evaluated by flow cytometry. Cell migration was monitored by in vitro wound healing and transwell migration assays. To induce fibroblastoid transformation, RPE were treated with recombinant human transforming growth factor (TGF)-β1 (10 ng/mL), and Western blot and immunocytochemical analyses were performed. The effect on RPE-collagen gel lattice contraction was determined by gel contraction assay. Bortezomib treatment decreased RPE population in a concentration-dependent manner and flow cytometry revealed that these effects were due to G2/M phase cell-cycle arrest. Bortezomib (20 nM) significantly inhibited RPE migration. Moreover, bortezomib treatment also significantly impeded TGF-β1-induced transdifferentiation of RPE. The effects on proliferation, migration, and EMT were mediated by regulation of NF-κB signaling pathway. Bortezomib prevented RPE-collagen gel lattice contraction. Bortezomib suppressed myofibroblastic transformation of RPE, and also inhibited RPE-collagen gel matrix contraction. Thus, bortezomib may be a candidate of putative therapeutic agent for management of fibrotic retinal diseases. 망막색소상피세포의 상피-간엽 변형에 있어 nuclear factor-kappa B (NF-κB)의 관련성이 보고되어 있다. 이에 본 연구에서는 프로테아좀(proteasome) 억제제인 bortezomib을 이용하여 망막색소상피세포의 증식과 이동 및 상피-간엽 변형의 억제효과에 대해 알아보고자 하였다. Bortezomib에 의한 세포 증식의 변화를 WST-8 분석법을 통해 확인하였고, 유동세포분석법을 통하여 bortezomib의 세포주기에 대한 효과를 확인하였다. 시험관내(in vitro) 상처치유 분석법 및 트랜스웰 이동 분석법을 통하여 망막색소상피세포의 이동에 대한 영향을 분석하였다. Transforming growth factor-β1 (TGF-β1)으로 상피-간엽 변형을 유발시킨 후, bortezomib을 처리한 후 상피-간엽 변형에 관여하는 다양한 표지자들의 발현변화를 분석하기 위하여 웨스턴 단백분석과 면역세포화학분석을 시행하였다. 또한, 막 수축에 대한 억제 효과를 알아보기 위하여 겔 수축분석(gel contraction assay)을 시행하였다. 20 nM농도의 bortezomib은 망막색소상피세포의 증식을 현저히 감소시키며 이는 G2/M기에서 세포주기를 정지시키는 것으로 확인되었다. Bortezomib은 망막색소상피세포의 이동과 TGF-β1으로 야기되는 상피-간엽 변형을 효과적으로 억제하였으며, 상피-간엽변형에 의한 막 수축도 효과적으로 억제하였다. Bortezomib은 망막색소상피세포에서 발현되는 NF-κB 세포신호전달기전을 하향조절 시킴으로써 망막색소상피세포가 근육아세포로 변형되는 것을 억제시키고 이를 통하여 망막색소상피세포-콜라겐 겔의 수축을 효과적으로 억제시켰다. 본 연구의 결과는 bortezomib이 섬유증식과 관련된 다양한 망막질환들에 있어서 치료제로서의 가능성을 갖고 있음을 제시하는 것이라 할 수 있겠다.