Broadening the Role of Osteocalcin in Leydig Cells

The demonstration that bone is an endocrine organ has been objectively an important recent advance in skeleton biology. Indeed, it has changed the type of questions asked in this field, because it has immediately connected bone biology to the rest of the body and to many complex physiological processes (1). Moreover, the fact that, via the hormone it produces, osteocalcin, bones regulate biological functions as diverse and unrelated to each other as glucose metabolism (2), male fertility in mice and humans (3, 4), and brain development and cognitive functions (5) raises a myriad of new questions that apply to various degrees to each of these functions. For instance, what is/are the receptor(s) used by osteocalcin to regulate each of these functions? What is the number and what are the identities of all the cellular processes regulated by osteocalcin in its known target cells? In addition, and this is a question that is specific to osteocalcin, because this protein is subjected to a posttranslational modification, which form of osteocalcin is acting as a hormone? De Toni et al (6) published in this issue of Endocrinology a focused, concise, and elegant study that manages to address all the aforementioned questions and to extend their reach to humans. They did so by focusing on one novel function exerted by osteocalcin in Leydig cells of the testis. It had been shown before that the uncarboxylated form of osteocalcin, but not the carboxylated one, favors expression of most but not all genes necessary for testosterone biosynthesis in Leydig cells. This occurs, in the mouse, after the binding of uncarboxylated osteocalcin to its specific receptor, a G-protein coupled receptor (GPCR) called Gprc6a (3). After the study of patients with peripheral testicular failure, these findings were subsequently extended to humans by showing that in at least 2 unrelated patients, the disease may be caused by a dominant negative mutation in GPRC6A (4). What De Toni et al did in this paper was to study whether the expression of another gene expressed in Leydig cells, Cyp2r1 (6, 7), which encodes an enzyme necessary for the 25 hydroxylation of vitamin D, is regulated by osteocalcin. The expression of this gene was not known to be under the control of osteocalcin in Leydig cells until this work. The authors first showed that, as it is the case for testosterone biosynthesis, it is the uncarboxylated form of osteocalcin that is regulating Cyp2r1 expression in a Leydig cell line and thereby the hydroxylation of vitamin D. They then went on to verify that this function of uncarboxylated osteocalcin occurs after its binding to its bona fide receptor Gprc6a (2, 3). Last but not least, De Toni et al showed that the ratio of uncarboxylated osteocalcin over total osteocalcin is a predictor of 25-hydroxy (OH) vitamin D circulating levels in humans, and in so doing, they add another dimension to their work. A major difference between the work of De Toni et al and the previous work describing the regulation of testosterone biosynthesis by osteocalcin is that unlike what was shown before for the regulation of testosterone biosynthesis (2), the authors failed to see a stimulation of cAMP production after stimulation of the cell line that they used by uncarboxylated osteocalcin. Of course, one cannot exclude a priori the possibility that osteocalcin uses different intracellular signaling pathways to regulate testosterone biosynthesis on the one hand and hydroxylation of vitamin D on the other hand. However, it seems unlikely that it would be the case. Moreover, the data incriminating ERK phosphorylation after osteocalcin treatment in this cell line are not the most convincing of the paper. There are alternative and more likely explanations for this apparent discrepancy. For instance, the authors are using a cell line, and it is difficult to know, given the information provided, whether this cell line contains or not all the machinery necessary to produce cAMP that is present in primary Leydig cells. A second and even more likely explanation for this apparent discrepancy may simply be that, as shown by Oury et al (3), the concentration of osteocalcin used by De Toni et al in their experiments does not really result in an increase in cAMP production in primary Leydig cells (see Ref. 6 and figure 5D therein). The most likely reason for that absence of effect of osteocalcin is the desensitization of the osteocalcin receptor that takes place when high doses of the hormone are used (3). This clever study is another example added to the very long and growing list of studies showing how animal models can reveal important biological processes. It makes another point. More often than not, novel physiological processes can be uncovered first in model organisms before they can be verified and extended to humans, where the investigation often can only be correlative. In that respect, this study, because of the way it was constructed, is an excellent example of how asking experimentally a focused question can reveal insight in biological processes that are affected in humans diseases like obesity in the present case. This being said, and as all excellent studies do, the work of De Toni et al comes with its lot of new challenges and new questions. A first question that now needs to be addressed is to determine the relevance in vivo of these findings obtained in a cell line to physiological and pathological situations in humans. This is particularly important, because circulating levels of 25OH vitamin D3 are similar in females and males, but osteocalcin does not act in the female gonads. Moreover, it will be important to determine whether the circulating levels of 25OH vitamin D3 decrease in men castrated for prostate cancer (8). A second question will be to determine what the respective contribution of LH and osteocalcin is in vivo in the regulation of the hydroxylation of the vitamin D in Leydig cells? A broader question prompted by this study is to know whether osteocalcin regulates more than testosterone biosynthesis and hydroxylation of the vitamin D3 in Leydig cells, in other words, how many distinct cellular pathways are influenced by bone in Leydig cells of the testis? It is likely that at least some answers to these questions will come in the near future. Even more broadly, this study raises the question of the identity of all the function of osteocalcin. This is now the most important challenge the field is facing.