The Role of PBR/TSPO in Steroid Biosynthesis Challenged

The events that regulate the rapid synthesis of steroid hormones in response to trophic hormone stimulation of the steroidogenic cells have been the ongoing subject of intense interest for several decades. Much of the early work performed in this area determined that the acute regulation of steroid hormone biosynthesis required the rapid, de novo synthesis of a protein(s) whose function appeared to be involved in mediating the delivery of cholesterol, the substrate for all steroid hormones, from the outer mitochondrial membrane to the inner mitochondrial membrane. The existence of a protein that was necessary for intramitochondrial transfer of cholesterol was first postulated by James Ferguson in 1963 (1). This transfer is an absolute requirement for steroid biosynthesis, because the cholesterol side-chain cleavage enzyme system that converts cholesterol to pregnenolone, the first steroid synthesized, resides on the inner side of the inner mitochondrial membrane. As such, the hydrophobic cholesterol substrate is unable to traverse the aqueous intermembrane space and reach the inner mitochondrial membrane through diffusion. The next 3 decades saw a focused interest in determining the identity of this putative regulator protein (2–13). These studies gave rise to a list of the characteristics that the putative protein regulator appeared to possess, including those described above. Later work introduced other putative regulator proteins, the sterol carrier protein 2 (14), the steroidogenesis activator polypeptide (15, 16), the peripheral benzodiazepine receptor (PBR) (17), and the steroidogenic acute regulatory protein (StAR) (18). Although space limitations do not allow for a critical evaluation of the characteristics of each of the candidates that have been put forth as the putative regulator protein over the years, these candidates have been described in an earlier review (19). One of the protein regulator candidates, PBR, recently underwent a change in its name and is now called the translocator protein (TSPO) (20). Early work on PBR/TSPO indicated that it was found in high concentrations in steroidogenic cells and that it was localized to the outer mitochondrial membrane, both positive and encouraging characteristics for a putative regulator (17). Specific studies on PBR/TSPO indicated that treatment of steroidogenic cells with ligands to PBR/TSPO were able to stimulate steroid biosynthesis in those cells (21–32), that inhibition of PBR/TSPO expression in cultured Leydig tumor cells resulted in an inhibition of steroid biosynthesis (33, 34), and that stable transfection of the cells with a vector containing the PBR cDNA was able to rescue steroidogenesis (34). More recent studies have claimed that PBR/TSPO is a component of a multiprotein complex termed the transduceosome that initiates cholesterol transfer to the mitochondria in a hormone-stimulated manner (35). Following this report, the presence of an 800-kDa protein complex, termed the metabolon, consisting of PBR/TSPO, the voltage-dependent anion channel, the ATPase family AAA domain-containing protein 3, and the cytochrome P450scc (side-chain cleavage) enzyme (CYP11A1), that was required for hormone-induced cholesterol transport to the P450scc enzyme was described (36). As a result of these studies, it was concluded that PBR/TSPO “is an indispensable element of the steroidogenic machinery, where it mediates the delivery of the substrate cholesterol to the inner mitochondrial side chain cleavage cytochrome P450” (34). In vivo studies on the role of PBR/TSPO were hampered by the fact that an attempt to generate PBR/TSPO knockout mice was reported to result in early embryonic lethality (37). The very important manuscript by Morohaku et al (42) proposes a serious challenge to the concept that PBR/TSPO is an indispensable requirement for cholesterol transfer to the P450scc enzyme and subsequent steroid biosynthesis. In this study, the authors first generated mice that contained TSPO floxed alleles (TSPOfl/fl). They then used these animals to make conditional testicular cell-specific knockout mice (TSPOcΔ/Δ) by crossing the floxed female mice with anti-Mullerian hormone receptor type II cre/+ (Amhr2cre/+) males and then backcrossing to generate the conditional deletion. The colony was maintained by breeding the TSPOfl/fl females with the TSPOcΔ/Δ males to provide pups of both sexes for subsequent experiments. These matings resulted in the females giving birth to viable pups and having the same litter sizes as nonknockout animals. Baseline testosterone levels in TSPOcΔ/Δ males were unaffected, and even their response to human chorionic gonadotropin stimulation was similar to TSPOfl/fl cohorts. The very first thing to be considered is that this is a conditional knockout specific for testicular Leydig cells as opposed to the global knockout that resulted in embryonic lethality in a previous report (38). Therefore, the effects studied by Morohaku et al (38) are specific for PBR/TSPO activity in the steroidogenic function of Leydig cells without affecting other tissues and survival. Thus, this precise approach has allowed examination of PBR/TSPO function in a setting that is more physiological than any method used in the past. Given these results, the reason underlying embryonic mortality of the global PBR/TSPO knockout becomes a question of interest. Details on the recombination strategy used to generate the global knockout were never published, and the exact stage of embryonic mortality was not reported. If PBR/TSPO is required for vital functions involved in embryonic development separate from steroid hormone biosynthesis, it may be necessary to revisit the global knockout to gain functional understanding of this protein. The observation that the testis-specific PBR/TSPO knockout animals are completely fertile and have normal levels of circulating plasma testosterone is also a difficult problem to reconcile. In light of the many reports, as cited above, that the presence of PBR/TSPO is an absolute requirement for steroid biosynthesis, it is difficult to understand how these animals make normal amounts of testosterone and are completely fertile. Although it would be difficult to imagine, could the source of the circulating testosterone be from another steroidogenic tissue, such as the adrenal, acting in compensation for a lack of testicular testosterone? This is yet another reason why studying the phenotype of the PBR/TSPO global knockout animal may be important. If this is not a viable approach, it will also be possible to use conditional knockouts in other steroidogenic organs/tissues to corroborate the present findings. It is further difficult to understand, in view of earlier published results indicating that knockdown of PBR/TSPO in cultured Leydig tumor cells resulted in a decrease in the expression of the StAR protein and its processing from a 37-kDa precursor to its mature 30-kDa form, why there is no decrease in testicular StAR in the present studies. What might be the explanations for these observed differences between the earlier work in tumor Leydig cells and the testis-specific knockout mice described here? Perhaps the difference lies in the fact that tumor Leydig cells in culture behave much differently than Leydig cells found in the testes of intact, wild-type animals. If so, it is imperative to determine why these differences are seen. Do the Leydig cells in the PBR/TSPO conditional knockout animals respond to the loss of PBR/TSPO with some as yet unidentified compensatory mechanism? If this were the case, it is important to know why the cultured Leydig tumor cells do not display this same compensatory mechanism when PBR/TSPO is knocked down. It may be beneficial to reexamine the PBR/TSPO knockdown experiments using both cultured Leydig tumor cells as well as primary cultured Leydig cells isolated from intact animals. In addition, the authors' laboratory could examine primary Leydig cells isolated from the conditional PBR/TSPO knockout animals to determine whether these cells respond identically in culture to those in the intact animals. It is also important to note that PBR/TSPO is expressed in organs that are not involved in steroidogenesis. Even in reproductive tissues, prominent PBR/TSPO expression can be seen in several nonsteroidogenic cell types (38). Pathological overexpression of PBR/TSPO is also seen in several tumors and cell lines in which these cells do not have a direct connection to steroid hormone biosynthesis (39, 40). Moreover, the role of the PBR/TSPO binding partner, acyl-coenzyme A-binding protein (previously known as the diazepam binding inhibitor), that was initially claimed to be important for steroidogenesis (26) has come under renewed scrutiny, because it has been shown that acyl-coenzyme A-binding protein knockout mice do not have a steroidogenesis-associated phenotype (41). Although these observations can be considered as evidence that PBR/TSPO undoubtedly has functions unrelated to steroidogenesis, a quick perusal of the literature reveals that observations on the nonsteroidogenic functions of PBR/TSPO are considerably outnumbered by evidence in support of its role in steroidogenesis. There are many questions that arise as a result of the data presented by Morohaku et al (38). It will be important to resolve these discrepancies as quickly as possible, so that studies on the mechanism of the acute production of steroid hormones can proceed. If the intact animal is different than tumor cell lines, the wisdom of using such cell lines will come into question, given the fact that the intact animal represents a more physiological approach. Also, if PBR/TSPO is not required for steroid biosynthesis in intact animals, as has been widely reported for tumor cell lines, what is its function in the cell and, indeed, in the whole organism? Findings presented by Morohaku et al (38) are without question an alert that we may need to rebuild our understanding of how steroid hormone production is controlled. This work was supported by National Institutes of Health Grant HD-17481 and funds from the Robert A. Welch Foundation Grant B1-0028. Disclosure Summary: The author has nothing to disclose. peripheral benzodiazepine receptor steroidogenic acute regulatory protein translocator protein.