Influence of retinoic acid on TBX1 expression in myocardial cells induced by Shh and Fgf8

Abstract

The aim of this study was to explore the regulatory mechanism of retinoic acid (RA) on the TBX1 gene expression in myocardial cells. Ventricular cardiocytes were isolated from neonatal rats and cultured, and then treated with different concentrations of retinoic acid. The expression of Shh and Fgf8 at mRNA and protein levels in neonatal rat myocardial cells were measured by using RT-PCR and Western blot technique, respectively. There was basal expression of Shh and Fgf8 in the control group. When treated with 3×10^-7 mol/L RA, we observed that the expression of Shh mRNA and protein in neonatal rat myocardial cells were up-regulated by 1.51 (P<0.05) and 1.10 times (P<0.05), respectively. In comparison with the control group, under the concentration of 5×10^-7 mol/L RA, they were up-regulated by 2.21 (P<0.05) and 2.38 times (P<0.05) individually. Meanwhile, we could detect that the expression of Fgf8 mRNA and protein were up-regulated by 2.50 times (P<0.05) and 80% (P<0.05) separately compared with the control group after stimulation of 3×10^-7 mol/L RA, and they were up-regulated by 3.48 (P<0.05) and 2.04 times (P<0.05) individually after stimulation of 5×10^-7 mol/L RA. The results indicated that RA could induce the expression of Shh and Fgf8 in neonatal rat myocardial cells. At the same time, it has shown that Shh and Fgf8 were involved in the regulation process of RA on TBX1 expression.

Keywords

retinoic acid / Tbx1 protein / Shh protein / Fgf8 protein

Introduction

22q11.2 microdeletion syndrome is an extremely frequent chromosome microdeletion syndrome, including a series of abnormal phenotype syndromes. 22q11.2 microdeletion presents a high incidence rate in DiGeorge syndrome, conotruncal anomaly face syndrome and velo-cardio-facial syndrome [1,2]. Recently some researches have indicated that TBX1 plays a vital role in heart development and the pathogenesis of 22q11.2 microdeletion syndrome. The retinoic acid (RA) has an important regulation function in the heart development of the vertebrate as an endogenous signaling molecule. The regulating function mechanisms between RA and TBX1 are fairly complicated, including direct regulating function and indirect regulating function in which a multitude of regulatory factors participated [3-6]. Deep researches about these regulatory mechanisms would be of benefit to catch the damaged molecule mechanism in 22q11.2 microdeletion syndrome and the essence of the hereditary basis of the heart outflow tract in congenital heart disease.

Materials and methods

Animals

Fifty Sprague-Dawley neonate rats, aged 1-2 days, were purchased from the Laboratory Animal Center, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China.

Agents

The agents used in this study were as follows: new-born calf serum and high glucose Dulbecco’s minimum essential medium (DMEM) (Gibco, USA), Retinoic acid (Sigma, USA), type II collagenase and trizol (Invitrogen, USA), mice anti-rat α-sarcomeric actin monoclonal antibody and FITC-goat anti-mice fluorescence second antibody (Wuhan Boster Biological Technology, LTD, China), rabbit anti-rat Shh polyclonal antibody, rabbit anti-rat Fgf8 polyclonal antibody and rabbit anti-rat GAPDH polyclonal antibody (Santa Cruz, USA), corresponding second antibody and ECL kit (Pierce Company, USA), and protein electrophoresis molecular mass marker (Fermentas, USA).

Original myocardial cell culture

The Sprague-Dawley neonate rats, aged 1-2 days, were sterilized and the apex cordis tissues were obtained under a sterile operation. The apex cordis tissues were cut into pieces into a size of 1 mm × 1 mm × 1 mm. Sufficient quantum mixture of 0.08% trypsinase and 0.05% collagenase were added into the tissues and mixed sufficiently at 37°C in an aqueous bath, and the tissues were digested repeatedly, and then new-born calf serum DMEM culture fluid was added to stop digestion. The supernatant was discarded after centrifuge. Cell suspension was inoculated into culture flasks, and the cells were adhered to the inner wall for 80 min to remove cells except for myocardial cells as a result of different speeds. DMEM culture solution contained 10% new-born calf serum was applied to adjust cell concentration to 1 × 10⁹ cell/L, and then the cells were inoculated into 50 mL culture flask, and conventionally cultured for 48 h in CO₂ incubation cabinet.

Purifying myocardial cells by fluorescence-activated cell sorting (FACS)

Myocardial cells cultivated for 48 h were digested with 0.125% parenzyme. While the cell morphous had some alterations, new-born calf serum culture fluid was added to neutralize. After repetitive beats by pipette, a cell suspension was obtained. The cell suspension was centrifuged for 5 min at 1000 r/min, and then 2% bovine serum albumin/phosphate buffer solution (BSA·PBS) incubation solution was added and stirred to get single cell suspension. The cells were incubated at the use of mice anti-rat α-sarcomeric actin monoclonal antibody with a concentration of 1∶200 for 2 h at room temperature in incubation solution, and then centrifuged with cold phosphate buffered solution (PBS) at 1000 r/min, washed and repeated twice. Then, the cells were added with 2% BSA·PBS incubation solution and beat upon to get single cell suspension, incubated for 45 min at room temperature in incubation solution, and incubated at the use of FITC-Goat anti-mice antibody with a concentration of 1∶50, and then centrifuged with cold PBS at 1000 r/min, washed and repeated twice. The incubated cells were suspended in 2% BSA·PBS incubation solution again, and then separated quickly by FACS. The concentration of the separated cells was adjusted to 5 × 10⁸ cells/L by DMEM cultivated solution, inoculated into six-well plates, and then conventionally cultivated for 48 h in the CO₂ incubation cabinet.

Grouping and experiments

Grouping

After being separated and cultivated for 48 h, the myocardial cells were treated with RA of different concentrations. The 5 groups were as follows: Group 1: cultured cells; Group 2: cultured cells added with 0.1% dimethyl sulphoxide (DMSO); Group 3: cultured cells added with 0.1% DMSO and 1 × 10^-7 mol/L RA; Group 4: cultured cells added with 0.1% DMSO and 3 × 10^-7 mol/L RA; Group 5: cultured cells added with 0.1% DMSO and 5 × 10^-7 mol/L RA. The cells in each group were incubated for 8 h and then prepared for experiment.

mRNA extraction

According to the grouping, after the ending phase of stimulation, Trizol was added to extract the cell total RNA. RNA was dissolved with 30 μL RNase-free water. The concentration was determined by ultraviolet spectrophotometer, and the quality of RNA was detected by agarose gel electrophoresis.

Reverse transcription polymerase chain reaction (RT-PCR)

The RT-PCR kit was purchased from Takara Biotechnology (Dalian, China) Co., Ltd. The detection indices: all-actin as internal reference; the target genes: Shh, Fgf8. All primers were designed by Primer5.0, and synthesized by the Shanghai Biotechnology Co., Ltd (China). The details of primer length and reaction condition are shown in Table 1. The PCR products were managed through 1.5% agarose gel electrophoresis, photographed, and then electrophoresis band was analyzed by the gel imaging analytical system. The absorbance of each objective gene and all-actin amplification products were determined.

Protein extraction

According to the grouping, after the ending phase of stimulation, the cell layer was washed three times by cold PBS. 50 μL protein lysate/(5 × 10⁶ cells) (protein lysate containing 0.5 mol/L Tris-HCl (pH 8.0), 0.15 mol/L sodium chloride, 0.02% sodium azide, 0.1% Polyacrylamide, 100 mg/L phenylmethylsulphonyl fluoride, 1 mg/L Aprotinin, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate) was added to extract the total cell protein. The protein extract was centrifuged for 20 min at 12000 r/min at 4°C. Supernatant was separated and the concentration of protein was determined by ultraviolet spectrophotometer using the Bradford method, multi-packed and kept at -70°C.

Detection of objective genes by immunoblotting

The extraction of above-mentioned cell protein was managed through sodium dodecyl sulfate polyacrylamide gel electrophoresis. The electrophoresis voltage was 200 V. After electrophoretic separation, the protein was electrotransferred on the nitrocellulose filter and placed in PBS full of 5% defatted milk powder to seal for 3 h, and added with Shh (1∶1000), Fgf8 (1∶500) polyclonal antibody to incubate at 4°C, and kept overnight. The nitrocellulose filter was washed by 0.3% Tween-PBS three times, for 15 min each time. After washing, the nitrocellulose filter was placed in IgG marked by horse radish peroxidase, incubated for 2 h on the rocking bed at room temperature. It was washed three times by 0.3% Tween-PBS, for 15 min each time. Next rabbit anti-rat GAPDH (1∶500) polyclonal antibody was applied to perform the same procedures, conducted as above comparison. Enhanced chemiluminescence (ECL) was applied to develop an image for antigen-antibody compound, and the integrated absorbance (IA) of protein strap was analyzed by GELW4D software (IA = average absorbance × area). Relative level of target protein was presented as IA_{target protein}/IA_GAPDH.

Data processing

All data was processed by SPSS13.0 statistics software, and presented as \overline{x}\pm s. The comparison between multitude means was through one-way ANOVA. The comparison between two means was through t-test. A P<0.05 was considered statistically significant.

Results

Cultivating myocardial cell

Neonate rat myocardial cells just after separation were round and uniform in size. After cultivation for 48 h, most cells adhered and integrated well into functional synplasm, with pulsation synchronization (Fig. 1).

Fluorescence activated cell sorting

Myocardial cells cultivated for 48 h were marked with mice anti-rat α-sarcomeric actin monoclonal antibody and corresponding second antibody, and then, green fluorescence cells were separated by flow cytometry. Separation efficiency was 78.5% (Fig. 2).

mRNA expression of myocardial cells

There was a little of Shh and Fgf8 expression in the control group. When treated with 3 × 10^-7 and 5 × 10^-7 mol/L RA, we observed that the expression of Shh mRNA in neonatal rat myocardial cell, had an increase of 1.51-fold (P<0.05) and 2.21-fold (P<0.05), respectively, in comparison with that in the control group. Meanwhile, we detected that the expression of Fgf8 mRNA had a 2.50-fold (P<0.05) and 3.48-fold rise (P<0.05) separately compared with that in the control group after stimulation of 3 × 10^-7 and 5 × 10^-7 mol/L RA (Fig. 3).

Protein expression of myocardial cells

There was a basal expression of Shh and Fgf8 in the control group. When treated with 3 × 10^-7 mol/L and 5 × 10^-7 mol/L RA, we observed that the expression of Shh protein in neonatal rat myocardial cells had an increase of 1.10-fold (P<0.05) and 2.38-fold (P<0.05), respectively, in comparison with that in the control group. Meanwhile, we detected that the expression of Fgf8 protein had a rise of 80% (P<0.05) and 2.04-fold (P<0.05) separately, compared with that in the control group after stimulation of 3 × 10^-7 mol/L and 5 × 10^-7 mol/L RA (Fig. 4).

Discussion

22q11.2 microdeletion syndrome is an extremely frequent chromosome microdeletion syndrome. Recently, following the development of genetics and reproductive science, we found that TBX1 was significant in 22q11.2 microdeletion syndrome discovered by targeted mutagenesis and transgenic animal experiments. At present, international and domestic scholars are investigating the genetics of 22q11.2 microdeletion syndrome, and have proven that TBX1 gene single copy depletion in mouse may be the major reason. TBX1 controls the transcription and expression of a series of downstream genes and impacts corresponding developmental phenotype as a transcription factor related with development [7-9]. RA can change the transcription of specific genes through the combination with intranuclear RA receptor as endogenous signaling molecule, and plays an extremely important regulating function in vertebrate heart development. RA and TBX1 are both absolutely necessary in pharyngeal arch development. The homeostatic damage of RA expression could further influence pharyngeal tissue and its differentiated tissue, which also result in similar phenotype like 22q11.2 microdeletion syndrome. Some research has demonstrated that RA at some concentration could promote mRNA expression of myocardium TBX1 gene, which presented a dose-dependent effect [10]. At present, the mechanism of upstream and downstream genes of TBX1 and their relationships are not clear. Recently, some scholars [11] thought that Shh might be the downstream gene of RA, and it could induce TBX1 expression. Shh is a signaling conduction factor closely related with growth and development, and plays an important role in embryonic development of many organs, such as four limbs, lung, nervous system and cardiovascular system. Recent research [12] indicated that Shh could regulate TBX1 expression during pharyngeal arch development process. Shh played an important role in the development of the arterial arcades. Moreover, Shh has shown a regulation to mesoblast and mesochymal growth and development genes of the pharyngeal portion [12]. The downstream gene induced by TBX1 is unknown yet. Researches have indicated that fibroblast growth factor expressed in the endoderm of embryo pharyngeal arch and directly transmitted information to mesenchymal cell originated from neural crest cells, which might be the downstream regulation factor of TBX1 [13,14]. Researches found that except TBX1, Fgf8 was the only one gene which has been proven to have the tissue-specific dependent function in the development of the fourth pharyngeal aortic arch, especially in the pharyngeal portion ectoblast. Mouse mutant embryo with Fgf8 mutant has heart defects similar to 22q11.2 microdeletion syndrome [15]. The penetrance of mutant aortic arch defect containing TBX1+/- and Fgf8+/- double heterozygote depletion simultaneously was significantly higher than that of the mutant having the single heterozygote depletion [16]. The expression of Fgf8 decreased in TBX1 defect embryo, thus it could be supposed that TBX1 could directly regulate Fgf8 in pharyngeal portion ectoblast [17]. Our research confirmed the mechanism of RA on TBX1 expression.

At present, myocardial cells could not be purified strictly in many experiments. It is well known that the contaminate of myofibroblasts, endothelial cells and vascular smooth muscle cells are unavoidable in the conventional culture of myocardial cells. In order to avoid those influential factors, in our research, fluorescence-activated cell sorting separation was selected to isolate and purify myocardial cells, the anti α-sarcomeric actin monoclonal antibody specificity aimed directly at striped muscle actin. The striped muscle actin only presented at skeletal muscle cells and myocardial cells. Of course, there were no skeletal muscle cells in heart. Therefore, anti α-sarcomeric actin monoclonal antibody could specifically separate high purified myocardial cells from heart mixed cells, and then the effectiveness mechanism of RA on TBX1 was studied by using RT-PCR and Western blot technique, in purified myocardial cells.

In this research, all-actin and GAPDH were selected as internal parameters to observe the difference in the expression of Shh and Fgf8 genes at mRNA and protein levels in the RA stimulated group and the control group. Quantitative analysis results indicated that Shh and Fgf8 has shown a lower expression in the control group. After stimulation with RA, mRNA and the protein of Shh and Fgf8 in cells were increased greatly. The expressions of Shh and Fgf8 were enhanced with the increasing RA concentration, which might happen due to the following mechanisms. One was RA might have a direct regulating function to TBX1. When the bird embryo and P19 cell was detected by reverse transcription real-time quantitation PCR, it was found that RA had a regulating function on TBX1 both in vivo and in vitro [18]. Another, the effect of RA on TBX1 was through an indirect and special way. And this indirect way means RA could cause the quantitative alteration of other transcription factors, which might influence the expression of TBX1. These factors included Shh gene. Shh could regulate Foxa2 and Foxc2 situs genes of pharyngeal mesoblast and head desmohemoblast. Foxc1 situs gene and Foxc2 situs gene were abundant expressed transcription factors in head mesoderm and aortic arch surrounding mesoderm. Foxa2, Foxc1 and Foxc2 could combine and activate transcription of TBX1 through cis-acting element, thus switch on the transcription factor of TBX1 downstream, which was Fgf8 [11, 12]. So the following regulating line might exist: RA→ Shh → (Foxc1, Foxc2, Foxa2) → TBX1 → Fgf8. Above conclusions were achieved from animal experiments and the definitive results would be confirmed through the study on the decoration of the endogenous gene element in vivo.

In conclusion, we have demonstrated that the regulating effect of RA on myocardial cell TBX1 is complicated, including direct regulation and indirect regulation, and a multitude of regulatory factors were involved [19, 20].Through the research of the complicated network relationship between upstream and downstream genes, the pathogenetics on the congenital malformation of heart will be further identified, and a novel rationale for the clinical diagnosis and treatment of 22q11.2 microdeletion syndrome will be provided.

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