Sex-dependent expression of CYP2C11 in spleen, thymus and bone marrow regulated by growth hormone

Abstract

CYP2C11, the most commonly expressed isoform of cytochrome P450 in male rat liver, was measured in spleen, thymus and bone marrow by quantitative real-time PCR and enhanced Western blotting. CYP2C11 concentrations in the lymphoid tissues were a fraction of that observed in liver, but like the liver, were sexually dimorphic (M>F) with mRNA and protein levels in agreement. Although the response to hypophysectomy varied according to tissue and sex, expression levels of CYP2C11 in all measured tissues remained greater in males. Further differences in CYP2C11 expression between liver and lymphoid tissue were observed following restoration of the circulating masculine growth hormone profile in hypophysectomized rats. In contrast to the liver where the renaturalized growth hormone profile elevated CYP2C11 expression in both sexes, the response was opposite in spleen and thymus with isoform concentrations declining in both sexes. Lastly, the divergent response of CYP2C11 between the liver and immune system was examined in cultured splenocytes exposed to different mitogens. In contrast to the dramatic depletion of CYP2C11 reported in proliferating hepatocytes, mitogen-stimulation resulted in a significant elevation in splenocyte CYP2C11 expression. In summary, we report for the first time that thymus, spleen and bone marrow express, albeit nominal, sex-dependent levels of CYP2C11 (M>F) whose regulation appears to be under some hormonal control, but very different from that of the hepatic isoform.

Figure 1

CYP2C11 Protein Levels in Hematopoietic Tissues. Western blot analysis was performed using microsomes from liver (2 μg protein), thymus, spleen and whole cell lysate of bone marrow (40 μg protein) obtained from intact and hypophysectomized (HYPOX) male and female rats. Bands are representative of an n≥5. ND, not detected.

Figure 2

Plasma Levels of Circulating Growth Hormone Obtained from Individual Undisturbed Catheterized Intact and Hypophysectomized (HYPOX) Episodic Rat Growth Hormone (rGH)-Treated Male and Female Rats. Serial blood samples were obtained from vehicle-infused intact and rGH-infused HYPOX rats after the 6^th intra-atrial injection of 40 μg of rGH/kg b. wt. Plasma was collected from the intact rats for 7 consecutive hours to allow the rGH peaks to be aligned with the peaks of the infused HYPOX rats. Similar findings were obtained from at least 5 additional rats in each treatment group.

Figure 3

Hepatic, Thymic and Splenic CYP2C11 mRNA from Hypophysectomized (HYPOX) and Growth Hormone (GH)-Restored Male and Female Rats. The physiologic masculine episodic GH profile was restored to HYPOX male and female rats by infusing rat GH (40 μg/kg b. wt.) via an indwelling atrial catheter attached to an external pumping apparatus, as a 3 min pulse every 4 hr for 14 pulses. Control rats received GH-diluent. CYP2C11 mRNA was determined by semi-quantitative PCR. Values are presented as a percentage of CYP2C11 mRNA with the highest value in each panel arbitrarily designated 100%. Each data point is a mean ± SD of at least 5 animals for each treatment. * p<0.01, when comparing males and females with the same treatment. ^† p<0.01, when comparing GH effect in the same sex.

Figure 4

Response of Splenic CYP2C11 mRNA Exposed to Various Mitogens. Hypophysectomized male splenocytes were harvested and either analyzed immediately before plating (Zero Time Cells) or cultured for 4 days in complete media containing either phytohemagglutinin (PHA), concanavalen A (CONA), bacterial lipopolysaccharide (LPS) or diluent. CYP2C11 mRNA was determined by semi-quantitative PCR. Sufficient viable cells were isolated from a single spleen for all treatments presented in the figure. Values are presented as a percentage of CYP2C11 mRNA in splenocytes from Zero Time Cells arbitrarily designated 100%. Each data point is the mean ± SD of at least 5 animals for each treatment. * P<0.01, when compared to Zero Time Cells. ^† p<0.01, when compared to cultures exposed to Diluent.