Microarray and K-means cluster analyses of genes regulated by TRH and by thyroid hormone in TRH-deficient pituitary.

<p>RNA samples from pituitaries of wild-type, TRHKO (TRH−/−), and euthyroid TRHKO mice with thryroid hormone replacement (TRH−/−+T4) were subjected to a whole-genome microarray using Affymetrix Mouse Gene chip 320 2.0 Array. After exclusion of absent signals (less than 100 signals), 10445 out of 45102 genes expressed in the TRHKO pituitary were subjected to the cluster analysis using a K-means algorithm. Hierarchical cluster analysis was carried out using a computer program, Genowiz™ 3.2. A) Genes were divided into ten hierarchical clusters according to changes and profiles among three groups, wild-type (WT signal), TRH−/− (Homo signal), and TRH−/− + T4 (T4 signal). B) In addition to the TSHβ genes, cluster 5 contained the Egr-1, NR4A1, Glt2, and Tmsb10 genes. C) mRNA levels of other anterior pituitary hormones including POMC, GH, LHβ, FSHβ, and prolactin (PRL) were not altered among the three groups, suggesting the expression to be TRH-independent. D) Profiles of the NR4A1, NR4A2, Egr-1, Pit1 and GATA2 genes are depicted. Expression of NR4A1, NR4A2 and Egr-1 might be TRH-dependent. E) Confirmation of results of the microarray analysis of NR4A1 mRNA levels in the TRH-deficient pituitary by real-time PCR. TRHKO mice (TRH−/−, n = 3) were supplemented with thyroid hormone by the daily injection of T4 (TRH−/−+T4, n = 3) and with TRH by 1 mg/kg body weight/day with an Alzet pomp for 7 days (TRH−/−+TRH n = 3). Total RNA obtained from these pituitaries was subjected to real-time PCR. The NR4A1 mRNA level in the TRHKO pituitary was decreased to about 25% of that of the wild-type. The decreased NR4A1mRNA level was not altered by thyroid hormone replacement, but completely reversed to the normal level by TRH administration, suggesting expression of NR4A1 mRNA in the pituitary to be TRH-, but not thyroid hormone-dependent.