Identification of UDP-Glucuronosyltransferase 2B15 (UGT2B15) as a Target for IGF1 and Insulin Action

Abstract

Normal growth and development in mammals are tightly controlled by numerous genetic factors and metabolic conditions. The growth hormone (GH)-insulin-like growth factor-1 (IGF1) hormonal axis is a key player in the regulation of these processes. Dysregulation of the GH-IGF1 endocrine system is linked to a number of pathologies, ranging from growth deficits to cancer. Laron syndrome (LS) is a type of dwarfism that results from mutation of the GH receptor (GHR) gene, leading to GH resistance and short stature as well as a number of metabolic abnormalities. Of major clinical relevance, epidemiological studies have shown that LS patients do not develop cancer. While the mechanisms associated with cancer protection in LS have not yet been elucidated, genomic analyses have identified a series of metabolic genes that are over-represented in LS patients. We hypothesized that these genes might constitute novel targets for IGF1 action. With a fold-change of 11.09, UDP-glucuronosyltransferase 2B15 (UGT2B15) was the top up-regulated gene in LS. The UGT2B15 gene codes for an enzyme that converts xenobiotic substances into lipophilic compounds and thereby facilitates their clearance from the body. We investigated the regulation of UGT2B15 gene expression by IGF1 and insulin. Both hormones inhibited UGT2B15 mRNA levels in endometrial and breast cancer cell lines. Regulation of UGT2B15 protein levels by IGF1/insulin, however, was more complex and not always correlated with mRNA levels. Furthermore, UGT2B15 expression was dependent on p53 status. Thus, UGT2B15 mRNA levels were higher in cell lines expressing a wild-type p53 compared to cells containing a mutated p53. Animal studies confirmed an inverse correlation between UGT2B15 and p53 levels. In summary, increased UGT2B15 levels in LS might confer upon patient's protection from genotoxic damage.

Keywords: Laron syndrome; UGT2B15; growth hormone receptor; insulin-like growth factor-1 (IGF1); p53.

Figure 1

Gene expression of UGT2B15 mRNA in LS patients. Total RNA was obtained from four LS- and four healthy control-derived EBV-immortalized lymphoblastoid cell lines. UGT2B15 mRNA levels were measured by RT-QPCR, as described in Section 2. Results are expressed as UGT2B15 mRNA levels normalized to β-actin mRNA values. A value of 1 was assigned to the UGT2B15 mRNA levels in control cells. Bars denote mean ± SEM. * p < 0.01 vs. control cells.

Figure 2

Expression of UGT2B15 in endometrial cancer cell lines. (A) Total RNA was obtained from confluent USPC-1 and USPC-2 endometrial cancer cell lines and UGT2B15 mRNA levels were measured by RT-QPCR. A value of 1 was assigned to the UGT2B15 mRNA levels in USPC-1 cells. * p < 0.01 vs. USPC-1 cells. (B) Total protein was obtained from confluent USPC-1 and USPC-2 cell lines and UGT2B15, IGF1R, INSR, and p53 protein levels were measured by Western blots. HSP70 levels were assessed as a loading control.

Figure 3

Regulation of UGT2B15 gene expression by IGF1 and insulin. USPC-1 (A) and USPC-2 (B) cells were treated with IGF1 or insulin (50 ng/mL) for 24 h, after which total RNA was prepared and UGT2B15 mRNA levels were measured by RT-QPCR. A value of 1 was given to the UGT2B15 mRNA levels in control, untreated cells. * p < 0.01 vs. respective control cells. (C,D) Western blot analysis of hormone-treated USPC-1 and USPC-2 cells. Cells were treated with IGF1 or insulin for 24-h, after which total protein was prepared and UGT2B15 levels were measured by Western blots. Bar graphs represent UGT2B15 levels normalized to the corresponding HSP70 values. Values on the y-axis represent arbitrary units of absorbance. A value of 100% was given to control, untreated cells.

Figure 4

Expression of UGT2B15 in breast cancer cell lines. (A) Total RNA was obtained from confluent T47D and MCF7 cells and UGT2B15 mRNA levels were measured by RT-QPCR. A value of 1 was assigned to the UGT2B15 mRNA levels in T47D cells. * p < 0.01 vs. T47D cells. (B) Total protein was obtained from confluent T47D and MCF7 cell lines and UGT2B15, IGF1R, INSR, and p53 protein levels were measured by Western blots. Relative protein levels are expressed as protein levels normalized to the corresponding HSP70 value. Results of a representative experiment are shown, repeated three times with similar results.

Figure 5

Regulation of UGT2B15 gene expression by IGF1 and insulin in breast cancer cell lines. MCF7 (A) and T47D (B) cells were treated with IGF1 or insulin (50 ng/mL) for 24 h, after which total RNA was prepared and UGT2B15 mRNA levels were measured by RT-QPCR. A value of 1 was given to the UGT2B15 mRNA levels in control, untreated cells. * p < 0.01 vs. respective control cells. (C,D) MCF7 and T47D cells were treated with IGF1 or insulin for 24 h, after which total protein was prepared and UGT2B15 and p53 levels were measured by Western blots. Bar graphs represent UGT2B15 levels normalized to the corresponding HSP70 values. A value of 100% was given to control, untreated cells.

Figure 6

Effect of IGF1R and INSR inhibition on UGT2B15 gene expression. (A) MCF7 cells were treated with the selective IGF1R inhibitor AEW541 (10 mM) for 48 h (or left untreated, C), after which cells were harvested, total protein was prepared, and IGF1R, UGT2B15, and p53 levels were measured by Western blots. HSP70 levels were measured as a loading control. The bar graph denotes UGT2B15 and IGF1R levels in control (solid bars) and AEW541 treated cells (striped bars). (B) T47D cells were treated with the INSR inhibitor S961 (100 nM and 1 mM) for 2 h. Cells were then harvested and levels of phospho- and total-INSR, UGT2B15, and p53 levels were measured by Western blots. A value of 100% was given to control, untreated cells. * p < 0.01.

Figure 7

Effect of UGT2B15 abrogation on IGF1R signaling and cellular proliferation. MCF7 (A,B) and T47D (C,D) were treated with siRNA against UGT2B15 (or NT for control purposes) for 72-h (MCF7) or 96 h (T47D). At the end of the incubation period, cells were harvested, and the levels of IGF1R, INSR, UGT2B15, phospho- and total- AKT and ERK1/2, and p53 were measured by Western blots. HSP70 levels were measured as a loading control. Relative protein levels are expressed as protein levels normalized to the corresponding HSP70 value. Results of a typical experiment are presented. For cell proliferation measurements, cells were treated with siRNA against UGT2B15 (or NT siRNA) for 72 h (MCF7) or 96 h (T47D). Cells were counted using a cell counter. A value of 100% was given to the cell number in NT-treated (control) cells. * p < 0.01 vs. NT-treated cells.

Figure 8

Physical interactions between UGT2B15 and p53. T47D cells were lysed and immunoprecipitated with anti-p53. Precipitates were electrophoresed through 10% SDS-PAGE, blotted onto nitrocellulose filters, and immunoblotted with anti-UGT2B15 or anti-p53. IP, immunoprecipitation; WB, Western blotting; TL, total lysate. Results of a typical experiment repeated twice with similar results are shown.

Figure 9

In vivo analysis of interactions between UGT2B15 and p53. Whole skin tissue was obtained from p53-KO and wild-type mice and processed as described in Materials and Methods. Levels of UGT2B15 and p53 were measured by Western blots. GAPDH served as a loading control. Bars represent the mean ± SD of two independent samples. * p < 0.01 vs. p53 wild-type mice.