**2. Steroidogenesis in testicular Leydig cells**

quinone ring and a geranylgeranyl group (four isoprene units) as a side chain (**Figure 1**). MK‐ 4 is not commonly synthesized by bacteria and is instead alkylated from menadione, which is supplemented in animal feeds to increase vitamin K levels. In most organs, MK‐4 is converted from dietary vitamin K1 and other menaquinones [1, 2] via a process catalyzed by UbiA prenyltransferase domain containing protein 1 [3]. Furthermore, MK‐4 is prescribed as a

**Figure 1.** Chemical structure of MK‐4. MK‐4 has a 2‐methyl‐1,4‐naphthoquinone ring and a geranylgeranyl group (four

Vitamin K is a well‐known nutrient required for blood coagulation and bone metabolism. In recent years, novel functions of vitamin K against inflammation [5, 6], tumors [7–9], and ligand of the nuclear receptor PXR (also known as SXR) [10, 11] have been reported. These findings suggest the beneficial role of vitamin K, including MK‐4, in several biological processes. In rodents, MK‐4 is distributed throughout the body and is observed in high quantity in the liver, bone, brain, pancreas, and reproductive organs, even when animals are fed a low MK‐4 diet (**Figure 2**) [12–15]. However, the role of MK‐4 in these organs has not been well characterized. This chapter focuses on the functional effects of MK‐4 on steroidogenesis in testicular Ley‐

**Figure 2.** Vitamin K contents in rat tissues. Male Wistar rats were fed an AIN‐93G diet for three weeks. Tissue levels of vitamin K and MK‐4 were determined by fluorescent HPLC (reproduced with permission from Shirakawa et al. [14]).

therapeutic agent for osteoporosis and to prevent fractures in Japan [4].

isoprene units) as a side chain.

190 Vitamin K2 - Vital for Health and Wellbeing

dig cells.

The major function of testicular Leydig cells is to produce testosterone in response to the pituitary luteinizing hormone (LH) as shown in **Figure 3**. The LH receptor (LHR) affects the 3′,5′‐cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway by associating with G proteins containing the cytoplasmic Gαs subunit. Production and secretion of testosterone in Leydig cells are tightly regulated by intracellular cAMP, a common secondary messenger. The formation and degradation of intracellular cAMP are under the control of adenylate cyclase (AC) and cyclic nucleotide phosphodiesterase (PDE), respectively. A rise in intracellular cAMP levels activates PKA, which stimulates downstream steroidogenic proteins. Steroidogenic acute regulatory (StAR) protein transports cholesterol to the inner mitochon‐ drial membrane of these cells to initiate steroidogenesis. Cytochrome P450scc (also known as CYP11A), a cholesterol side‐chain cleavage enzyme, catalyzes a cascade of reactions that convert cholesterol to the steroid hormone precursor pregnenolone, which is then transformed into testosterone [16]. Both StAR and CYP11A constitute rate‐limiting steps in the overall steroidogenesis of testosterone from cholesterol.

**Figure 3.** Steroidogenic pathway in testicular Leydig cells. AC, adenylate cyclase; CREB, cAMP response element‐bind‐ ing protein; CYP11A, cholesterol side‐chain cleavage enzyme; G, G protein; HSD, hydroxysteroid dehydrogenase; LH, luteinizing hormone; LHR, LH receptor; Mito, mitochondria; PDE, cyclic nucleotide phosphodiesterase; PKA, protein kinase A; SER, smooth endoplasmic reticulum; StAR, steroidogenic acute regulatory protein.

Testosterone production in men is crucial for fetal development, sperm production, and the development of male secondary sex characteristics. Furthermore, increasing evidence shows that lowering testosterone production causes infertility and sexual dysfunction in men, including cases of late‐onset hypogonadism (LOH) [17, 18]. The current progressive aging of the population and the stress of modern life may also contribute to LOH, resulting in sexual dysfunction, muscle weakness, and even depression. Moreover, low testosterone levels can also predict the development of several diseases, such as type 2 diabetes and cardiovascular disease [19–21]. This issue could be addressed by identifying nutrients that may boost testosterone levels.

We also found a relationship between vitamin K and steroidogenesis in testis‐derived tumor cells. After incubation of two testis‐derived cell lines, mouse I‐10, and rat R2C, in the presence of MK‐4, we detected a dose‐dependent rise in secreted testosterone in culture medium [26]. In I‐10 cells, MK‐4, but not vitamin K1, led to increased levels of testosterone. We also found that menaquinone‐3 and menaquinone‐7 stimulated testosterone production in I‐10 cells (unpublished data). These results suggest that menaquinones, which have an unsaturated isoprenyl side chain, enhance testosterone production. In I‐10 cells, the production of proges‐ terone, a testosterone precursor, increased in a dose‐dependent manner following treatment with MK‐4. This indicates that stimulation of steroidogenesis by MK‐4 occurs upstream of progesterone synthesis (**Figure 2**). To assess the effects of MK‐4 on the activation of PKA in I‐ 10 cells, a reporter gene assay was employed (**Figure 4**). The cAMP response element‐driven luciferase reporter plasmid was transfected into I‐10 cells. Accordingly, MK‐4 enhanced reporter activity relative to the control. Moreover, Western blot analysis revealed that MK‐4 increased the expressions of Cyp11a, as well as phosphorylation levels of PKA and the cAMP response element‐binding protein (CREB) in I‐10 cells. In contrast, the increase in testosterone level induced by MK‐4 was completely abolished by treatment with the PKA inhibitor H89 [26]. These results indicate that, unlike vitamin K1, MK‐4 significantly stimulates testosterone

Menaquinone‐4 Enhances Steroidogenesis in Testis Derived Tumor Cells Via the Elevation of cAMP Level

http://dx.doi.org/10.5772/63982

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**Figure 4.** PKA activation following treatment with MK‐4 in I‐10 cells. I‐10 cells were transfected with a plasmid bearing

Our findings were in accordance with previous studies by Ichikawa et al. [24], who showed that MK‐4, but not vitamin K1 or other vitamin K2 isoform, specifically induced mRNA levels of GDF15 and STC2, whose protein levels are regulated by PKA in human and mouse osteoblasts. However, Tsang and Kamei have reported that both vitamin K1 and MK‐4 promote nerve growth factor‐dependent outgrowth of neuronal cells, which were blocked in the

a cAMP response element fused to a luciferase reporter gene and then treated with MK‐4.

production and may play an important role in steroidogenesis.
