Pharmacological Regulation of the Cholesterol Transport Machinery in Steroidogenic Cells of the Testis (2015)
Abstract
Reduced serum testosterone (T), or hypogonadism, is estimated to affect about 5 million American men, including both aging and young men.
Low serum T has been linked to mood changes, worsening cognition, fatigue, depression, decreased lean body mass and bone mineral density, increased visceral fat, metabolic syndrome, decreased libido, and sexual dysfunction. Administering exogenous T, known as T-replacement therapy (TRT), reverses many of the symptoms of low T levels. However, this treatment can result in luteinizing hormone suppression which, in turn, can lead to reduced sperm numbers and infertility, making TRT inappropriate for men who wish to father children. Additionally, TRT may result in supraphysiologic T levels, skin irritation, and T transfer to others upon contact; and there may be an increased risk of prostate cancer and cardiovascular disease, particularly in aging men. Therefore, the development of alternative therapies for treating hypogonadism would be highly desirable. To do so requires a greater understanding of the series of steps leading to T formation and how they are regulated, and the identification of key steps that are amenable to pharmacological modulation so as to induce T production.
We review herein our current understanding of mechanisms underlying the pharmacological induction of T formation in hypogonadal testis.
Introduction
The administration of exogenous T by any means can suppress LH and thus result in reduced Leydig cell T formation and suppression of spermatogenesis. Indeed, contraception in men can be achieved by administering LH-suppressive T. Thus, the exogenous administration of T to ameliorate hypogonadism is inappropriate for men wishing to father children (Carruthers, 2009; Huhtaniemi & Forti, 2011; Perheentupa & Huhtaniemi, 2009; Riggs et al., 1982).
There are methods in use that increase serum T without T administration, including hCG treatment in the case of men with secondary hypogonadism. In individuals with primary hypogonadism, aromatase inhibitors can increase T-to-estradiol ratios, particularly in men with severe infertility, but often this approach is ineffective (Perheentupa & Huhtaniemi, 2009).
Increasing intratesticular and serum T by stimulating the Leydig cells themselves could have great advantages. Such an approach should not elicit significant fluctuations in T levels because T formation would be regulated at least in part by the negative feedback of T on LH. Nor would there be T transfer to others via contact.
Fertility should be preserved, not suppressed, because the hypothalamic-pituitary axis should not be shut down as with exogenous T. Indeed, the local stimulation of Leydig cell T production by the use of drug ligands that target proteins involved in cholesterol import into mitochondria, the rate-determining step in T formation, might actually support or enhance spermatogenesis because intratesticular T levels should increase, not decrease. This advantage could be of great benefit to the many young men with primary hypogonadism who wish to father children. If successful, T replacement through the administration of drugs targeting proteins involved in T formation could constitute a paradigm shift in the treatment of hypogonadal men.
In summary, the design of new therapies that increase intratesticular bioactive androgen levels without affecting the hypothalamic-pituitary axis would be of great benefit to numerous patients. This approach requires an understanding of the series of steps leading to T formation and how they are regulated, and the identification of key steps amenable to pharmacological modulation to induce T.
4 STEROID BIOSYNTHESIS
Steroidogenic cells are defined by their ability to convert the precursor cholesterol to pregnenolone in the mitochondrial matrix through the function of the cytochrome P450 side-chain cleavage enzyme (CYP11A1) (Jefcoate, 2002), which is a member of a large family of P450 enzymes. Seven members of this family are targeted to mitochondria and the remaining 50 members to the endoplasmic reticulum (ER). Six of the members of this family are involved in steroidogenesis.
Mitochondrial CYP11A1 converts cholesterol to pregnenolone, with the latter, then converted to progesterone by 3β-hydroxysteroid dehydrogenase (3βHSD; HSD3B) present in both the mitochondria and ER. CYP17A1, present in the ER, possesses both 17α-hydroxylase and 17,20-lyase activities. In the gonads of men, CYP17A1 converts progesterone to dehydroepiandrosterone which is further metabolized to T by 17-βHSD (Fig. 1).
Figure 1 Summary of the steroidogenic pathway in the testis. The steroid hormone biosynthesis pathway in the Leydig cells of the testis is shown in this schematic representation. Cholesterol is the sole precursor of all steroids and it is through several enzymatic reactions in mitochondria, cytoplasm, and ER that different types of steroids are synthesized due to the presence and activity of specific enzymes. P5, pregnenolone; P4, progesterone; T, testosterone.
The rate-limiting step in steroidogenesis is the import of cholesterol into mitochondria to become accessible to CYP11A1. By limiting access of the hydrophobic cholesterol molecule to CYP11A1, steroidogenic cells are able to control the amount of steroids they produce (Rone, Fan, & Papadopoulos, 2009). Steroid hormones are continuously synthesized. However, in response to increased circulating peptide hormones (LH in the case of Leydig cells), the rate of steroid hormone synthesis is greatly increased. LH BINDS ITS COGNATE RECEPTORS ON THE SURFACE OF LEYDIG CELLS AND STIMULATES INTRACELLULAR SIGNALING CASCADES, OF WHICH THE cAMP PATHWAY, THROUGH PROTEIN KINASE A (PKA), IS THE MOST PROMINENT. ACUTELY, THESE EVENTS STIMULATE THE IMPORT OF CHOLESTEROL TO THE OMM WHERE IT IS CONVERTED TO PREGNENOLONE BY CYP11A1 (Jefcoate, 2002; Papadopoulos, Liu, & Culty, 2007). IN ADDITION, HORMONAL AND cAMP STIMULATION OF STEROIDOGENIC CELLS ARE IMPORTANT FOR THE CHRONIC REGULATION OF STEROIDOGENESIS, AS CONTINUED STIMULATION IS NECESSARY TO ENSURE PROPER EXPRESSION LEVELS OF STEROIDOGENIC PROTEINS AND ENZYMES AS WELL AS STEROIDOGENIC METABOLIC FLUX (Simpson & Waterman, 1988).
The mitochondrion is a double-membrane organelle with an aqueous intermembrane space (IMS) between OMM and IMM, where CYP11A1 resides. Cholesterol molecules are hydrophobic.
Thus, the movement of cholesterol across the aqueous microenvironment of the mitochondria requires the involvement of intracellular machinery (Mesmin & Maxfield, 2009). Indeed, steroidogenic cells possess a multicomponent protein machine, the transduceosome, an ensemble of cytoplasmic and resident mitochondrial proteins that receive hormonal signals and is involved in the translocation of cholesterol across the IMS at contact sites between the OMM and IMM (Papadopoulos et al., 2007; Rone, Liu, et al., 2009) (Fig. 2). The transduceosome OMM proteins were identified as the 30-kDa voltage-dependent anion channel 1 (VDAC1) and the 18-kDa translocator protein TSPO, a high-affinity drug- and cholesterol-binding protein previously named the peripheral-type benzodiazepine receptor (McEnery, Snowman, Trifiletti, & Snyder, 1992; Papadopoulos et al., 2006).
In addition to the mitochondrial proteins making up core structural and enzymatic components of the transduceosome, cytoplasmic proteins are instrumental in regulating its assembly and cholesterol transport activity. Hormonal stimulation was found to promote the clustering of TSPO, which was correlated with steroidogenesis and could be suppressed by the PKA inhibitor H-89 (Boujrad, Gaillard, Garnier, & Papadopoulos, 1994). This suggests an active role for PKA in the assembly of the transduceosome. Yeast two-hybrid screening for additional cellular partners of TSPO yielded the acyl-CoAbinding domain family protein ACBD3/PAP7 (Li, Degenhardt, et al., 2001), which serves to scaffold the cytosolic PKA-RI subunits to the transduceosome (Li, Degenhardt, et al., 2001; Liu, Rone, & Papadopoulos, 2006). An additional ACBD protein family member, ACBD1, participates in transduceosome function. Originally identified through its ability to displace benzodiazepine bound to GABA receptor sites in neurons (Costa & Guidotti, 1991), and hence originally named the diazepam-binding inhibitor, ACBD1 acts on TSPO and stimulates steroidogenesis (Boujrad et al., 1994; Papadopoulos, Berkovich, Krueger, Costa, & Guidotti, 1991).
While gene expression of the transduceosome components has been the focus of numerous studies, much less is known about mechanisms through which transduceosome components are targeted to their proper locations in the mitochondria, a factor critical to their function in steroidogenesis (Black, Harikrishna, Szklarz, & Miller, 1994).
4.1 Mitochondrial protein import and chaperones
4.2 Transduceosome
4.2.1 Translocator protein
4.2.2 Voltage-dependent anion channel 1
4.2.3 Steroidogenic acute regulatory protein
4.2.4 Protein kinase A
4.2.5 Acetyl CoA-binding domain 3
4.3 Metabolon
4.3.1 Cholesterol side-chain cleavage cytochrome P450 (CYP11A1)
4.3.2 AAA+ ATPase, ATAD3
5. CAN SERUM TESTOSTERONE LEVELS BE INCREASED BY STIMULATING THE LEYDIG CELLS THEMSELVES?
As indicated above, there are good reasons to increase serum T levels in hypogonadal men, but doing so by TRT is less than ideal for both young and aging men.
There are methods to increase serum T without T administration, including administering LH (or hCG) for men with secondary hypogonadism. In men with primary hypogonadism, aromatase inhibitors can increase T-to-estradiol ratios, but often this approach is ineffective (Perheentupa & Huhtaniemi, 2009). Increasing intratesticular and serum T by stimulating the Leydig cells themselves could have great advantages in part because doing so would be expected to result in the physiological regulation of the T that is produced by its negative feedback on LH. This is, in contrast, to essentially flooding the system with T, as with exogenous T administration. Stimulating the Leydig cells should not elicit significant fluctuations in T levels, and nor there be T transfer to others via contact. Fertility should be preserved, not suppressed, because the hypothalamic-pituitary axis should not be shut down as with exogenous T.
Indeed, the local stimulation of Leydig cell T production might actually enhance spermatogenesis because intratesticular T levels should increase, not decrease. Moreover, Leydig cells produce more than just T, and so their stimulation might produce Leydig cell products in addition to T that might have effects on spermatogenesis. With an understanding of the series of steps leading to T formation derived from numerous studies conducted over the years and the identification of key steps that have been shown to be amenable to pharmacological modulation so as to induce T production, it now might be possible to stimulate Leydig cells to produce additional T as a means by which to increase serum T levels.
5.1 TSPO drug ligands
5.2 14-3-3γ and ε proteins
6. CONCLUSION
Cholesterol is the main precursor of T. Its import into mitochondria is the rate-limiting step in steroidogenesis. After a decade of intensive research, it is now clear that cholesterol import into the mitochondria in response to hormonal stimulation is carried out through a multiprotein complex called transduceosome, and not by individual proteins (Midzak, Rone, Aghazadeh, Culty, & Papadopoulos, 2011; Papadopoulos & Miller, 2012; Rone et al., 2012).
In search of mechanisms inducing T production in the Leydig cell as an alternative to TRT, components of the transduceosome were identified to be critical for T formation and “druggable” candidates. Indeed, TSPO and 14-3-3ε–VDAC1 interactions were shown to be viable targets which, when activated, result in increased T formation both in vitro and in vivo, thus providing new means for the treatment of androgen deficiency in hypogonadal men.
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