Expression Of ABCG2 Transporter And Analysis Of Wnt Signaling Mechanism During Regulated Proliferation And Differentiation Of Tracheal Stem Cells

IntroductionA stem cell can be defined as any proliferated cell which retains a high capacity for self—renewal and can give birth to high differentiation daughter cells in determinate condition. Stem cells can be divided into two groups according to their origin: embryonic stem cells and adult stem cells. Recent years more and more scientists have focused on the adult stem cells. Several adult stem cells have been discovered, such as hemo-poietic stem cell, bone marrow stem cell, neural stem cell, muscle stem cell, cornea stem cell, et al. Now many of them have been used for clinical treatment. However, stem cells in adult respiratory system remains largely unknown. We have developed an injury model of tracheal epithelium induced by 5 —FU in vitro successfully to observe the changes in tracheal epithelial stem cells, and characterized them primarily.Stem cells can exclude the Hoechst 33342 dye. Recent studies implicated that expression of ABCG2, the ATP binding cassette (ABC) transporter (i. e. breast cancer resistance protein 1, BCRP1), could mediate the SP phenotype. High expression of SP cells have been detected in various tissues from many species, including bone marrow, liver, muscle, breast epithelium and ES cells. Thus, the molecule ABCG2 that determines the SP phenotype may be potentially a common novel stem cellmarker, and contributes to isolating the stem cells from tissues. To explore the proliferation and differentiation of tracheal stem cells, we utilized the injury model induced by 5 —FU and examined changes expression of ABCG2 in tracheal epithelium during the process of regeneration was analyzed by indirect immunofluorescence and Wetern blot. Evidence is e-merging that the Wnt/j3 —catenin pathway may be important in regulating stem cell self — renewal and differentiation programs. Recent evidence suggests that the Wnt signaling pathway, along with its components, has been shown to be crucial in directing cell fate during embryo-genesis. This pathway also regulates proliferation and differentiation of primitive cells in adult tissues such as the gut, liver, hematopoietic system and skin. To explore the mechanism of involvement of the Wnt/j3 — catenin pathway during regulated growth of tracheal epithelial stem cells in rats, we identified changes in epithelial morphology, levels of Wntl, |3 — catenin, and cyclinDl mRNAs, and localization of (3 —catenin protein during rat tracheal epithelial regeneration using reverse transcriptase— polymerase chain reaction (RT —PCR) and indirect immunofluorescent techniques. Our findings indicate that the Wnt/p —catenin pathway plays a role in proliferation and differentiation of tracheal epithelial stem cells. Our studies will contribute to our understanding of the molecular mechanisms that regulate self—renewal and lineage—specific differentiation.Materials and Methods1. Preparation of Tracheal Epithelium Regeneration Model. Wistar rats of both sexes, about 200g in weight, were used for the experiments under the strict guidelines of the Animal Care Committee at the China Medical University. The tracheae were excised sterilely and cultured in 1:1 mix of Dulbecco's modified Eagle's medium and Ham's F—12 medium (DMEM/F12) containing 120 mg/ml 5—FU and 10% fetal bovine serum (FBS) for 12 h at 37°C. The tissues were then refreshed by culturing in DMEM/F12 containing 10% FBS. Tracheae were taken out at 0,3,6,9,12,24,48 h time points after removing 5 — FU. For RT—PCR, epithelium of some tracheae were stripped under anatomical microscope, snap — frozen and stored at —80°C until use. Some tracheae were fixed in 4. 5% formaldehyde, and made into paraffin— embedded tissue sections for he-matoxylin—eosin stain. Normal tracheae were also analyzed as controls.2. Isolation of Tracheal Epithelial Cells. Epithelial cells were isolated from fresh tracheae harvested at 0, 3, 6, 12, 24, 48 h time points and from normal tracheae. The cell suspensions were dropped onto the slides. After drying, the slides were soaked in 4% paraformaldehyde for 20min to fix the epithelial cells, and then stored at —20°C for immunofluores-cent observation.3. Indirect Immunofluorescence. Paraffin—embedded tissue sections were analyzed by indirect immunofluorescence measured polyclonal antibodies against ABCG2. TRITC—labeled anti — goat antibody as a secondary antibody were used. Experiments were also done on trachea epithelial cells smear. Primary antibodie was anti — j3 — catenin . Secondary antibody used was the same one to Paraffin— embedded tissue sections. Nuclei were counterstained with 1 /ig/ml DAPI in PBS. Negative controls were obtained by replacing the first antibody with PBS. Sections were viewed on an Olympus BX51FL Eclipse epi — fluorescence microscope. Digital images were obtained on a matched—Olympus camera and the Op-timas image acquisition software.4. Western blot Analysis. Fresh normal tracheas and tracheal tissues taken on 0,6,12,24,48 hours after removement of 5 —FU were analyzed. Samples containing 20 (ig protein were subjected to electrophoresis on 10% polyacrylamide gels. Proteins were transferred electrophoretically onto polyvinylidene fluoride (PVDF) membranes. After blocking, blots were probed with antibody against ABCG2. A secondary horseradish per-oxidase—conjucted antigoat antibody was applied for lh. Immunoreactive proteins were visualized after treatment of membranes with DAB kit.5. RT—PCR. Total RNA was extracted from tissue samples harvested previously. With TRIzol? reagent cDNA were prepared according tostandard methods. The following primers were used: Wntl (304bp) ? forward: 5V-AGG TGA AAG GGC AAG GAA-3\ reverse: 5V-CTG GCA GAC AAG AGG AGT GA-3y;j3 -catenin (5OObp): forward: 5V-GCG CTC CCC TCA GAT GGT GTC-3\ reverse: 5^-ACG ATG GCC GGC TTG TTG C-3^;cyclinDl (42Obp) : forward: 5*-TGG CGT TTG GAA GTA GGG-3\ reverse: 5^-GAG CGG CGG CAA GAA TGT -3*;13 -actin (587bp): forward: 5'-CCA AGG CCA ACC GCG AGA AGA TGA C-3\ reverse: 5V-AGG GTA CAT GGT GGT GCC GCC AGA C-3\ |3 — actin served as an endogenous control. PCR conditions: predenatur-ation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 50°C (Wntl and cyclinDl), 52eC (|3 —catenin) or 58°C (J3—actin) for 30 s and extension at 72°C for 1 min, 35 cycles. For negative controls, the PCR reaction was performed without prior reverse transcription. Amplified cDNA was visualized by ethidium bromide staining on 2% agarose gels on a gel scanner.Result1. After treatment with 5 — FU for 12h, the tracheal epithelium shed and cells in GO with naked nuclei were seen sparsely on the basement membrane, and ABCG2 positive cells were seen among them. 3h after the removal of 5 — FU, the tracheal rings were covered with flattened epithelium. ABCG2 positive cells increased slightly on the meantime. 6h after the removal of 5 — FU, With more flattened epithelial cells appeared, more ABCG2 expression were observed after the removal of 5 —FU . At 24h, most of the epithelial cells were cuboidal cells and merged into pieces, but the ABCG2 positive cells decreased obviously . Until 48h after theremoval of 5 — FU, only few positive cells could be seen with the pseudostratified mucociliary epithelium which was restored to its original mode similarly. There were no detectable ABCG2 —positive cells in normal tracheal epithelium2. There were different ABCG2 levels at different times after the re-moval of 5 — FU which in accordance with the change detected by immun-ofluorescene. ABCG2 was minimally detected after treatment with 5 —FU for 12h, reaching a maximal level at 6h after the removal of 5 —FU, and then decreased over time. Until 48h after the removal of 5 —FU, ABCG2 level was very low.3. Result of RT —PCR indicated that no detectable levels of Wntl and cyclinDl mRNAs were found in normal tracheal epithelium. Level of j3—catenin mRNA was shown to be low. Immediately following the removal of 5 —FU, level of Wntl mRNA increased at this time, but level of P — catenin mRNA decreased slightly. Level of Wntl mRNA was elevated maximally at 3h after the removal of 5 —FU with the appearance of flattened epithelial cells. Levels of |3—catenin and cyclinDl mRNAs reached maximally at 6 and 12h respectively. At 24h decreased levels of Wntl, |3 — catenin and cyclinDl mRNAs were observed. At 48h, there were no detectable levels of Wntl and cyclinDl mRNAs. Level of {3 —catenin mRNA decreased to less than normal level.4. Membrane location of (3—catenin was observed in normal epithelial cells. At 6 and 12h, (3 — catenin moved from cell membranes to cytoplasm and nuclei, shortly after the level of Wntl mRNA reached maximally. Then at 48h, (3 —catenin returned to its membrane location.Conclusions1. Observed the changes of the ABCG2 expression in tracheal epithelium during the process of tracheal epithelial regeneration in rats. The expression of ABCG2 is correlated to the number of tracheal stem cells, suggesting that ABCG2 may serve as a marker for isolating stem cells from tracheal epithelium.2. Our findings indicate that the Wnt/j3 — catenin pathway plays a role in proliferation and differentiation of tracheal epithelial stem cells. Our studies will contribute to our understanding of the molecular mechanisms that regulate self—renewal and lineage—specific differentiation.