We found that although Adxsi-CMV-PDX1 induced small islet-like cells, the transcription levels of and were significantly lower than those in Adxsi-CMV-PDX1/CMV-PAX4-transfected cells (Figure 7A, ?,7B7B). Open in a separate window Figure 7 Co-expression E7080 (Lenvatinib) of PAX4 and PDX1 in MSCs promotes the activation of and expression. was recovered. To remove EGFP, the parental pShuttle-EGFP-CMV vector was digested with gene coding sequence fragment was ligated to the pShuttle-CMV vector with T4 ligase, generating E7080 (Lenvatinib) a pShuttle-CMV-PDX1 vector. The target gene fragment was obtained from the pEGFP-N1-PAX4 vector with tests with SPSS 15.0 software. All data are expressed as the mean standard deviation. Results Restriction enzyme digestion analysis The pAdxsi-CMV-EGFP, pAdxsi-CMV-PDX1, and pAdxsi-CMV-PDX1/CMV-PAX4 plasmids were digested with and (Figure 7A). We found that although Adxsi-CMV-PDX1 induced small islet-like cells, the transcription levels of and were significantly lower than those in Adxsi-CMV-PDX1/CMV-PAX4-transfected cells (Figure 7A, ?,7B7B). Open in a separate window Figure 7 Co-expression of PAX4 and PDX1 in MSCs promotes the activation of and expression. A. RT-PCR analysis of and gene expression. and mRNA levels in the Adxsi-CMV-PDX1 group are markedly lower than those in the Adxsi-CMV-PDX1/CMV-PAX4 group. B. Results are expressed for the different groups as the ratio of or mRNA to -actin mRNA levels. Data represent the mean SEM; n = 3 in two different experiments. *< 0.001. The protein levels of GLUT2 and insulin as determined by Western blotting were E7080 (Lenvatinib) consistent with their respective mRNA levels (Figure 8A, ?,8B).8B). Indirect fluorescent analysis demonstrated that PAX4/MafA and insulin as well as MafA and C-peptide (C-P) were present in the same cell (Figure 8C). Thus, Adxsi-CMV-PDX1/CMV-PAX4-infected cells expressed proinsulin, which was further hydrolyzed into functional insulin and C-P. The GLUT2 expression levels in Adxsi-CMV-PDX1-infected MSCs were significantly lower than those in Adxsi-CMV-PDX1/CMV-PAX4-infected MSCs, suggesting that PAX4 enhanced the PDX1 induction of MSCs into islet-like cells. We hypothesized that the expression of MafA and PAX4 may play key roles in upregulating Nkx6.1 and other transcription factors in the induced cells. Thus, proinsulin, GLUT2, and related intracellular hydrolases are further increased in the induced MSCs, which ultimately differentiate into functional insulin-secreting cells (Figure 8D). Open in a separate window Figure 8 Co-expression of -cell function-related molecules in cells of the Adxsi-CMV-PDX1 and Adxsi-CMV-PDX1/CMV-PAX4 groups. A, B. Characterization of GLUT2, Insulin, PAX4, and PDX1 protein expression in Adxsi-CMV-PDX1/CMV-PAX4 and Adxsi-CMV-PDX1 group cells. C. Immunofluorescence detection f both insulin or C-peptide (C-P, red) and PAX4 or MafA (green) shows co-expression in the cells of Adxsi-CMV-PDX1/CMV-PAX4 group. D. The schematic diagram is a simplified model indicating that transcription factors play roles in -cell development. PAX4 is required for the terminal differentiation of -cell function. Glucose-stimulated insulin secretion and C-P levels The culture fluid levels of insulin and C-P following stimulation of cells with 12.0 mmol/L glucose were measured by ELISA. The medium from the Adxsi-CMV-PDX1/CMV-PAX4 group contained significantly higher concentrations of C-P and insulin than that from the Adxsi-CMV-PDX1 group under high glucose stimulation (Figure 9), indicating that PAX4 and PDX1 play an important role in the differentiation of MSCs into mature cells capable of insulin synthesis and release in response to high glucose stimulation. Open in a separate window Figure 9 Co-expression of PDX1 and PAX4 improves glucose-regulated insulin and C-peptide secretion and increases the expression of -cell-related genes in cells 30 days after Adxsi-CMV-PDX1/CMV-PAX4 infection. A. Insulin secretion levels at 5.0 and 12.0 mM glucose in response to Adxsi-CMV-PDX1 or Adxsi-CMV-PDX1/CMV-PAX4. B. C-peptide (C-P) secretion levels at 5.0 and 12.0 mM glucose in response to Adxsi-CMV-PDX1 or Adxsi-CMV-PDX1/CMV-PAX4. n = 3 in two different experiments. *< 0.001. Discussion PDX1 contains a number of transcriptional regulatory regions, such as the N-terminus transcriptional regulatory elements related to the functional structure of the GG and insulin A3 domains, and is a key gene necessary for the development of pancreatic endocrine cells [19-21]. Rabbit Polyclonal to SHP-1 However, this development requires additional molecules. PDX1 also combines with the GLUT2 TATA box, contributing to GLUT2 transcription [21,22]. Recent studies have shown that PDX1 induces pancreatic differentiation and development. activation by glucose stimulation is controlled by NeuroD1 at transcriptional and post-transcriptional levels [35]. Ngn3 is an important islet progenitor cell marker [36]. In the formation of the pancreas, all endocrine cells are derived from Ngn3+ cells, and Ngn3 initiates the differentiation of endocrine progenitor cells [36]. The binding of E7080 (Lenvatinib) the bHLH domain of Ngn3 with the upstream promoter of the E-box domain activates the transcription of and [36,37]. By contrast, the binding between Ngn3s own promoters can inhibit.