dihydroxycholesterol OHC was recently identified by classic
7α,25-dihydroxycholesterol (7α,25-OHC) was recently identified by classic analytical methods as a high-affinity ligand for EBI2 (Hannedouche et al., 2011; Liu et al., 2011). 7α,25-OHC was previously identified as an intermediate in the alternate pathway of hepatic bile dl 473 synthesis (Russell, 2003). The conversion of cholesterol into bile acids is accomplished in the liver through two multienzyme pathways, commonly referred to as the classic and alternate pathways of bile acid synthesis. Studies in gene-deficient mice revealed that the essential requirement for bile acids can be met through either of the two pathways, and thus that they serve compensatory roles in hepatic lipid metabolism (Russell, 2003). 7α,25-OHC is synthesized from cholesterol by CH25H-mediated hydroxylation at the 25 position, followed by CYP7B1-mediated hydroxylation at the 7α position (Russell, 2003) (Figure 1A). Unexpectedly for a protein that carries out a reaction related to bile acid synthesis, CH25H is poorly expressed in the liver but is abundant in a number of other tissues, suggesting the enzyme may function outside the liver (Lund et al., 1998; Russell, 2003). Recent studies have shown that CH25H is highly expressed in activated macrophages (Bauman et al., 2009; Diczfalusy et al., 2009; Park and Scott, 2010; Zou et al., 2011). Genetic deficiency in CH25H has been shown to cause a loss of EBI2 ligand generation in lymphoid organs (Hannedouche et al., 2011). Although macrophages are considered the most likely cells acting in lymphoid tissues to carry out the 25-hydroxylation reaction needed to generate EBI2 ligand (Hannedouche et al., 2011; Liu et al., 2011), their role in this process was not tested.
CYP7B1, a member of the cytochrome P450 enzyme family, is abundant in the liver, but Cyp7b1 transcripts have also been detected in a number of extrahepatic tissues (Stiles et al., 2009). In the kidney, CYP7B1 may contribute to de novo sterol synthesis (Li-Hawkins et al., 2000), and in the reproductive tract, the enzyme has a role in metabolizing androgens (Omoto et al., 2005). In a recent report, treatment with the nonspecific cytochrome P450 inhibitor, clotramizole, reduced 7α,25-OHC in mouse spleen (Liu et al., 2011). This study provides support for CYP7B1 functioning in 7α,25-OHC generation in the spleen, but indirect effects of the drug could not be excluded. Moreover, the cell types involved in 7α,25-OHC generation were not determined.
Hydroxylated sterols are further metabolized to 3-oxo,Δ4 intermediates during the process of bile acid synthesis by a microsomal 3β-hydroxy-Δ5-C27 steroid oxidoreductase (HSD3B7) (Russell, 2003) (Figure 1A). This enzyme catalyzes isomerization of the double bond from the 5 to the 4 position and the oxidation of the 3β-hydroxyl to a 3-oxo group (Russell, 2003). HSD3B7 only acts on C27 sterols with a 7α-hydroxyl group. Loss of HSD3B7 blocks the synthesis of the major biologically active forms of bile acids, resulting in vitamin deficiency and cholesterol malabsorption (Shea et al., 2007). Mice with this defect can by rescued by addition of a pan-vitamin supplement to the drinking water and a bile acid (cholic acid) to the diet (Shea et al., 2007). It is not yet known whether HSD3B7 acts in nonhepatic tissue on substrates such as 7α,25-OHC, nor whether 7α,25-OHC modification by HSD3B7 alters EBI2 ligand activity.
Discussion CYP7B1 and HSD3B7 are best defined for their roles in bile acid synthesis in the liver (Russell, 2003). Our studies show that these two enzymes, together with CH25H, are required in lymphoid organs for the generation of EBI2 ligand gradients that guide the movements of naive and activated B cells. We have established that HSD3B7-mediated modification of 7α,25-OHC inactivates EBI2 ligand activity. We demonstrated that CH25H, CYP7B1, and HSD3B7 constitute a metabolic pathway that is required in lymphoid stromal cells for establishing B cell-guiding 7α,25-OHC gradients. We also have provided evidence that FDCs are needed to maintain a low-EBI2-ligand zone in the follicle interior and that HSD3B7 restricts EBI2 ligand production by T zone DCs.