A microarray analysis of the temporal response of liver to methylprednisolone: a comparative analysis of two dosing regimens

Abstract

Microarray analyses were performed on livers from adrenalectomized male Wistar rats chronically infused with methylprednisolone (MPL) (0.3 mg/kg.h) using Alzet mini-osmotic pumps for periods ranging from 6 h to 7 d. Four control and 40 drug-treated animals were killed at 10 different times during drug infusion. Total RNA preparations from the livers of these animals were hybridized to 44 individual Affymetrix REA230A gene chips, generating data for 15,967 different probe sets for each chip. A series of three filters were applied sequentially. These filters were designed to eliminate probe sets that were not expressed in the tissue, were not regulated by the drug, or did not meet defined quality control standards. These filters eliminated 13,978 probe sets (87.5%) leaving a remainder of 1989 probe sets for further consideration. We previously described a similar dataset obtained from animals after administration of a single dose of MPL (50 mg/kg given iv). That study involved 16 time points over a 72-h period. A similar filtering schema applied to the single-bolus-dose dataset identified 1519 probe sets as being regulated by MPL. A comparison of datasets from the two different dosing regimens identified 358 genes that were regulated by MPL in response to both dosing regimens. Regulated genes were grouped into 13 categories, mainly on gene product function. The temporal profiles of these common genes were subjected to detailed scrutiny. Examination of temporal profiles demonstrates that current perspectives on the mechanism of glucocorticoid action cannot entirely explain the temporal profiles of these regulated genes.

Fig. 1

Gene tree representation of all probe sets (15,967) on individual Affymetrix R230A gene chips hybridized to total RNA prepared from livers taken from animals treated chronically with MPL (0.3 mg/kg·h) for periods ranging from 6 –168 h. The values for each individual probe set at each time point were normalized to the mean value of that probe set for time zero controls. The x-axis represents the 11 time points, including time zero controls. The y-axis presents the list of probe sets grouped by similarity using Pearson’s correlation. Yellow indicates no change from controls, red indicates probe sets with enhanced expression relative to controls, and blue indicates suppressed expression relative to controls.

Fig. 2

The top panel provides a magnification of five probe sets selected from Fig. 1 that show apparent enhanced regulation by MPL. Two of these probe sets represent the same gene (Glud1). The linear plots for both Glud1 probe sets are presented in the lower panel.

Fig. 3

Gene tree representation of probe sets remaining (1989) after filtering.

Fig. 4

MPL concentrations in rat plasma after chronic (left) and acute (right) administration of drug. MPL concentrations were determined by normal-phase HPLC analysis of plasma samples obtained from individual animals.

Fig. 5

Response profiles of two genes showing enhanced expression after acute and chronic MPL dosing. Ornithine decarboxylase (Odc1) expression is shown in the two left panels and exhibits a similar pattern of enhanced expression with acute and chronic dosing. Tryptophan 2,3-dioxygenase (Tdo2) is presented in the two right panels and exhibit dissimilar patterns with acute and chronic dosing.

Fig. 6

Response profiles of two genes showing reduced expression after acute and chronic MPL dosing. Progestin and adipoQ receptor family member VII (paqr 7) expression is shown in the two left panels and exhibits a similar pattern of reduced expression in the two dosing regimens. Kupffer cell receptor (Kclr), which exhibits dissimilar patterns of regulation with acute and chronic dosing, is presented in the two right panels.

Fig. 7

Response profiles of two genes showing complex regulation of expression after acute and chronic MPL dosing. G9/G1 switch gene 2 (GOS2) and Arginase (Arg1) both exhibit initial down-regulation followed by a period of enhanced regulation.

Fig. 8

Response profiles of two genes showing complex regulation of expression after acute and chronic MPL dosing. In this case, both heterogenous nuclear ribonucleoprotein methyltransferase (Hrm1L2) and nuclear receptor superfamily 0, group B member 2 (NROb2) show initial up-regulation followed by a later down-regulation.