CAMK2 Expression and Its Regulation on Testosterone Synthesis in Mouse Testis

Abstract

Leydig cells are testosterone synthesis cells in testes, a process tightly regulated by luteinizing hormone (LH) through the activation of steroidogenic enzymes such as steroidogenic acute regulatory protein (StAR) and 3-beta-hydroxy-Delta5-steroid dehydrogenase (3β-HSD). While calcium/calmodulin-dependent protein kinase 2 (CAMK2) is known to modulate diverse cellular processes, including hormone signaling, its role in testosterone production remains unclear. In this study, we investigated the expression and functions of CAMK2 in mouse testes, focusing on its potential involvement in testosterone synthesis. Our findings demonstrate that CAMK2 expression progressively increases from postnatal day 1 (PND 1) to adulthood. Pharmacological inhibition of CAMK2 with KN-62 markedly reduced serum testosterone levels and downregulated the expression of key steroidogenic enzymes, including StAR and 3β-HSD, at both mRNA and protein levels. In vitro experiments using primary Leydig cells further confirmed that CAMK2 inhibition suppressed testosterone production and steroidogenic enzyme expression, particularly after prolonged (12–24 h) KN-62 treatment. Additionally, CAMK2 expression was upregulated in response to LH stimulation, suggesting its involvement in LH-mediated signaling pathways, potentially through modulation of the epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase 1/2 (ERK1/2) cascade. These findings demonstrate that CAMK2 positively regulates testosterone synthesis in Leydig cells, likely via the EGFR/ERK1/2 cascade. The results of this study enhance our understanding of the regulation of testosterone synthesis and identifies CAMK2 as a potential therapeutic target for male reproductive endocrine disorders.

INTRODUCTION

Testosterone, the primary male sex hormone, plays a crucial role in male reproductive development, spermatogenesis, and the maintenance of secondary sexual characteristics. Testosterone deficiency (hypogonadism) is associated with infertility, metabolic syndrome, cardiovascular disease, and decreased bone mineral density.^1–3) Although luteinizing hormone (LH) analogs and testosterone replacement therapies are commonly used to treat hypogonadism, their long-term efficacy and side effects underscore the need for a deeper understanding of steroidogenic regulation.^4,5) It has thus been extensively studied about the mechanisms underlying testosterone production, and physiological and therapeutic relevance.

Testosterone is primarily synthesized in Leydig cells, located in the testicular interstitium.⁶⁾ Its production is tightly controlled by the hypothalamic–pituitary–gonadal (HPG) axis, with LH serving as the key regulator.^7,8) Upon binding to its receptor (LHR) on Leydig cells,^9,10) LH triggers cAMP production, initiating steroidogenesis.¹¹⁾ This process upregulates steroidogenic enzymes, including steroidogenic acute regulatory protein (StAR), CYP, family 11, subfamily a, polypeptide 1 (CYP11A1), 3-beta-hydroxy-Delta5-steroid dehydrogenase (3β-HSD), and CYP, family 17, subfamily a, polypeptide 1 (CYP17A1),^12–15) which collectively convert cholesterol into testosterone. Dysregulation of these enzymes contributes to disorders such as congenital adrenal hyperplasia and Leydig cell insufficiency.¹⁶⁾

The signaling pathways underlying LH-mediated testosterone synthesis and the molecular mechanisms have been extensively studied. Multiple signaling pathways, including PKA, PKC, mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR), and extracellular signal-regulated kinase (ERK)1/2, mediate LH-induced testosterone synthesis.^17–21) However, the role of calcium/calmodulin-dependent protein kinase 2 (CAMK2) in Leydig cell steroidogenesis remains poorly understood, despite its reported involvement in spermatogenesis and Sertoli cell function.²²⁾

CAMK2 is a multifunctional serine/threonine kinase with isoforms (α, β, γ, δ) that exhibit tissue-specific expression. Activated by calcium-calmodulin complexes, CAMK2 transduces calcium signals into cellular responses, influencing synaptic plasticity, gene transcription, and hormone secretion.^23–25) Its regulatory role in hormonal pathways, pharmacological modulation of CAMK2 (e.g., via KN-93 or CAMK2 overexpression) may offer therapeutic potential for endocrine disorders.²⁶⁾ Although CAMK2 influences testicular function,^22,27) its role in testosterone synthesis remains unexplored. Since CAMK2 interacts with pathways such as MAPK/ERK1/2, known regulators of steroidogenesis,²⁵⁾ we hypothesized that CAMK2 may mediate LH-induced testosterone production.

This study investigates the developmental expression of CAMK2 in developmental mouse testes and its functional role in testosterone synthesis using pharmacological inhibition in vivo and in vitro. Our findings reveal that CAMK2 is upregulated by LH and may act upstream of EGFR/ERK1/2 to regulate steroidogenesis. These results identify CAMK2 as a novel modulator of Leydig cell function and a potential target for treating male reproductive disorders.

MATERIALS AND METHODS

Animals and Treatment

The male mice of wild-type C57 strain were purchased from Laboratory Animal Center of Veterinary Medicine College in Yangzhou University. All mice were housed at 24 ± 2°C with relative humidity of 55 ± 15% under 12 h/12 h light–dark cycle. Experimental schemes in animal were approved by the Institutional Animal Care and Use Committee (IACUC) at Yangzhou University.

For KN-62 (CAMK2 inhibitor; HY-13290, MedChemExpress, Monmouth Junction, NJ, U.S.A.) injection, 8-week mice were anesthetized with 0.25% tribromoethanol (HY-B1372, MedChemExpress) at a dose of 200 mg/kg‧body weight. Following anesthesia, the testes were exposed and bilaterally injected with KN-62 (640 μg/kg‧body weight).^28,29) After 24 h, blood was collected by an eyeball method and stored overnight at 4°C to obtain serum. The mice were sacrificed by cervical fracture. Testes were collected and stored in liquid nitrogen for experiment.

Antibodies and Inhibitors

CAMK2γ antibody (12666-2-AP; Proteintech Group, Inc.; Wuhan, China), phospho-CAMK2β/γ/δ antibody (YP0781; ImmunoWay; San Jose, CA, U.S.A.), 3β-HSD antibody (sc-30820; Santa Cruz Biotechnology; Santa Cruz, CA, U.S.A.), StAR antibody (12225-1-AP; Proteintech Group, Inc.), GAPDH antibody (M20006; Abmart, Shanghai, China), Phospho-p44/42 MAPK(ERK1/2) antibody (9101, Cell Signaling Technology, Boston, MA, U.S.A.), ERK1/2 antibody (4695, Cell Signaling Technology), Phospho-EGFR Antibody (30277-1-AP; Proteintech Group, Inc.), EGFR Antibody (18986-1-AP; Proteintech Group, Inc.), RAF1 Antibody (12552; Cell Signaling Technology).

MK-2206 (AKT inhibitor, HY-108232, MedChemExpress), CH (PKC inhibitor; HY-12048, MedChemExpress), gefitinib (EGFR inhibitor; HY-50895, MedChemExpress), H89 (PKA inhibitor; HY-15979, MedChemExpress) and RAF709 (RAF1 inhibitor; HY-100510, MedChemExpress).

Isolation and Identification of Primary Leydig Cells

Primary Leydig cells (pLCs) were isolated from 8-week-old male mice (10–15 mice) euthanized by cervical dislocation. Testes were collected and transferred to sterile conditions, surrounding connective tissues were carefully removed under a stereomicroscope. Testes were washed twice with Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; D2906, Sigma-Aldrich, St. Louis, MO, U.S.A.). Tissue digestion was performed using 8 mL 0.5‰ collagenase I (C0130, Sigma-Aldrich) at 37°C for 15 min. Seminiferous tubules were removed by sequential filtration through 200 and 400-μm mesh filters. Then cell suspension was centrifuged at 1200 × g for 5 min. Erythrocyte lysis was performed using 5 mL red blood cell lysis buffer (R1010, Solarbio, Beijing, China) for 5 min, followed by centrifuging at 1200 × g for 5 min. The pellet was resuspended in DMEM/F12 and subjected to discontinuous Percoll (Sigma- P4937, Sigma-Aldrich) density gradient (60, 37, 26, and 21%) centrifugation at 3000 × g for 30 min. PLCs were collected from the 60–37% interface and cultured with DMEM/F12 supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco) and 1% (w/v) penicillin-streptomycin (516106, Sigma-Aldrich).

The cell purity was identified by immunofluorescence using 3β-HSD antibody (1 : 50; sc-30820, Santa Cruz Biotechnology, Dallas, TX, U.S.A.). After the cells adhere to culture dish, the pLCs were treated with cold methanol for 15 min and were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, T8787) in PBS for 20 min. After washing thoroughly with PBS, the pLCs were incubated in PBS with 10% donkey serum for 1 h followed by incubated with anti-3β-HSD antibody overnight at 4°C. The next day, the pLCs were incubated with donkey anti-goat IgG H&L (1 : 500; ab150129, Abcam, Cambridge, U.K.) at room temperature for 2 h. The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 : 10000; C1002, Beyotime Biotechnology, Shanghai, China) for 15 min. Finally, cells were mounted with 15 μL Anti-Fade Mounting Medium (E675011; Sangon Biotech, Shanghai, China). The images were photographed under a fluorescence microscope photograph system (Olympus, Tokyo, Japan).

Transient Transfections

Camk2g siRNA kit were purchased from GenePharma (Shanghai, China). Transient transfections were performed using Lipo8000 (Beyotime Biotechnology) according to the manufacturer’s recommendations. The sequences are listed in Table 1. Testosterone concentrations in cultured medium and genes expressions were detected at 36 h after transfection.

Table 1. siRNA Sequences Targeting Camk2g

siRNA	Sequences (5′-3′)
Negative control	Sense-UUCUCCGAACGUGUCACGUTT
	Antisense-ACGUGACACGUUCGGAGAATT
siCAMK2γ-1	Sense-CCCGAGAUCAUCAGAAACUTT
	Antisense-AGUUUCUGAUGAUCUCGGGTT
siCAMK2γ-2	Sense-GAGGAAAGAUCCCUAUGGATT
	Antisense-UCCAUAGGGAUCUUUCCUCTT
siCAMK2γ-3	Sense-CGUGGUACAUAAUGCUACATT
	Antisense-UGUAGCAUUAUGUACCACGTT
siCAMK2γ-4	Sense-GUGGCUCAAUGUCCACUAUTT
	Antisense-AUAGUGGACAUUGAGCCACTT

Immunohistochemistry

Tissues were fixed with 4% paraformaldehyde, then dehydrated by graded ethanol, followed by paraffin embedding, and cut into 5-μm sections in transverse directions. Immunohistochemistry (IHC) staining was performed as describedpreviously.³⁰⁾ Sections were dewaxed and rehydrated. After antigen retrieval by microwaving sections in 0.01 M sodium citrate buffer (pH 6.0), the sections were then incubated in 3% (v/v) H₂O₂. Non-specific binding sites were then blocked by 10% normal goat serum (Beyotime Biotechnology). CAMK2 antibody (1 : 200) diluted in phosphate-buffered saline (PBS) was added and incubated overnight at 4°C. After washing with PBS for 30 min, the sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1 : 200, Jackson Immuno Research Laboratories, Inc., West Grove, PA, U.S.A.) for 2 h at room temperature. Peroxidase activity was detected by diaminobenzidine (Zhongshan, Beijing, China) staining for less than 2 min.

Real-Time Quantitative PCR (RT-qPCR)

Total RNA of the uterus tissues was isolated using the TRIzol reagent (TaKaRa, Dalian, China), purified by DNase I and quantified by spectrophotometry. A total of 1 μg of purified total RNA was used as a template for cDNA synthesis using HiScript Reverse Transcriptase (Vazyme, Nanjing, China) according to the manufacturer’s instructions. RT-qPCR was performed using SYBR Green master mix (Vazyme) in the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, U.S.A.) and reactions were done in triplicate. RT-qPCR conditions were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative gene expressions were normalized to endogenous control Gapdh. All Primers listed in Table 2 were designed using NCBI.

Table 2. Primer Pair Sequences Used in RT-qPCR

Gene	Primer sequences (5′-3′)
Camk2a	F-CACCACCATTGAGGACGAAGA
	R-ACCCATCAGGTGGATGTGAG
Camk2b	F-TGGGATGAGGACCAACACAAG
	R-GAGTAACGGTGTCCCACTCA
Camk2d	F-AACGCTCTACTGTTGCCTCC
	R-CGCCCTTCAGTTTCCGTCTA
Camk2g	F-CACCGACGACTACCAGCTTT
	R-GGGCGGACAACTTCTTGGTA
3β-HSD	F-GAGGAGATCAGGGTCCTGG
	R-CTAGGATGGTCTGCCTGGG
Star	F-TCCCTCAAAGACCAAACTCAC
	R-AGTGGCTGGCGAACTCTATCT
Gapdh	F-GCTCACTGGCATGGCCTTCCGTG
	R-TGGAAGAGTGGGAGTTGCTGTTGA

Western Blot (WB)

The uterus tissues were lysed with RIPA buffer (P0013B, Beyotime Biotechnology, Shanghai, China) containing 1 mM phenylmethanesulfonyl fluoride (PMSF, Sangon Biotech). The protein concentration of each group was determined using the BCA assay reagent (CoWin Biosciences, Jiangsu, China) according to the manufacturer’s recommendations. Equal amounts of 50 μg proteins were electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and the bands were transferred to 0.45 μm polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, U.S.A.). The membrane was blocked with 5% (w/v) nonfat dry milk in 0.05 mol/L pH 7.4 Tris buffered saline (TBS) for 1 h and incubated with CAMK2 antibody (1 : 2000), phospho-CAMK2β/γ/δ antibody (1 : 2000), 3β-HSD antibody (1 : 1000), StAR antibody (1 : 1000), p-EGFR antibody (1 : 1000), EGFR antibody (1 : 1000), p-ERK1/2 antibody (1 : 2000), ERK1/2 antibody (1 : 2000), RAF1 antibody (1 : 1000) and GAPDH antibody (1 : 1000) overnight at 4°C. The PVDF membrane was then washed 3 times for 30 min in TBST (0.1% Tween-20 in TBS) and incubated for 2 h with horseradish peroxidase-conjugated goat anti-rabbit IgG or horseradish peroxidase-conjugated goat anti-mouse IgG (Zhongshan), horseradish peroxidase-conjugated Rabbit anti-Goat IgG (CSB-PA00220F1Rb; CUSABIO, Wuhan, China). After washing for 30 min with 3 changes of TBST, the membrane was treated with the ECL kit (Vazyme) and visualized by Tannon gel imager (Tanon, Shanghai, China). The intensity values pertaining to each group were normalized against the optical density of GAPDH corresponding to the same group.

Radioimmunoassay

Testes of different developmental stages were lysed in RIPA lysis buffer (200 mg/300 μL), supernatant was collected following sonication and centrifugation at 15000 × g for 30 min at 4°C. Cultured pLCs were incubated with KN-62 and the culture medium was collected. Both tissue lysates and culture medium were stored at –80°C for testosterone analysis. Experiments were performed at least three times. Testosterone was analyzed using radioimmunoassay (RIA; Beijing North Institute Biological Technology, Beijing, China).

Statistical Analysis

All data analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, U.S.A.). The results are expressed as the mean ± standard error of at least three independent experiments. Student t tests or one-way ANOVA were used to determine differences between groups. p-Values of 0.05, 0.01, or 0.001 were considered statistically significant and indicated with an asterisk (*, **, or ***) in each chart, respectively. Different superscript letters above the bars denote statistically significant differences (p < 0.05).

RESULTS

CAMK2 Expression in Mouse Testes

To investigate the expression patterns of CAMK2 isoforms during testicular development, we analyzed the mRNA levels of Camk2a, Camk2b, Camk2g, and Camk2d in mouse testes at postnatal days (PND) 1, 7, 14, 21, 30, and 60 using RT-qPCR. The results revealed distinct expression patterns among the isoforms. Camk2a mRNA levels were significantly lower at PND 7 compared with PND 1 and remained low from PND 14 to PND 60 (Fig. 1A). By contrast, Camk2b mRNA levels remained consistently low across all developmental stages, with no significant differences observed (Fig. 1A). Camk2g expression was low from PND1 to PND 14 but increased significantly at PND 21, PND 30, and PND 60 (Fig. 1A). Notably, Camk2d mRNA levels were markedly higher at PND 60 compared with earlier stages and were substantially more abundant than all other Camk2 subtypes at every time point examined (Fig. 1A).

Fig. 1. CAMK2 Expression in Mouse Testis and Leydig Cells

(A) RT-qPCR analysis of Camk2 mRNA expression in testes of male mice at different ages, n = 3. (B) Testosterone contents in testis measured by radioimmunoassay (RIA), n ≥ 3. (C, D) Represent Western blot and analysis of CAMK2γ protein levels in mouse testes at different ages, n = 3. (E) Immunohistochemical staining of CAMK2γ (brown) in paraffin-embedded testis sections, counterstained with hematoxylin (blue). Red arrows indicate Leydig cells. Bar = 50 μm. Data represent mean ± standard error of the mean (S.E.M.). Different superscript letters above the bars denote statistically significant differences (p < 0.05).

As the expression pattern of Camk2g mRNA paralleled the rise in testicular testosterone levels after birth (Fig. 1B), we further analyzed CAMK2γ protein expression. Western blot analysis confirmed that CAMK2γ protein levels increased progressively from PND 1 to PND 60, peaking at PND 60 (Figs. 1C, 1D). Immunohistochemical staining localized CAMK2γ to the cytoplasm of Leydig cells (Fig. 1E), suggesting a potential role for CAMK2 in regulating testosterone synthesis post-puberty.

CAMK2 Involves in Regulating Testosterone Synthesis in Mouse Testes

To determine whether CAMK2 is involved in regulating testosterone synthesis, 640 μg/kg‧body weight CAMK2 inhibitor (KN-62) was injected into the testes of adult mice.²⁸⁾ The mice were scarified and blood was collected at 24 h after injection. The level of phosphorylated CAMK2 and CAMK2γ protein was detected to analyze its kinase activity in the presence and absence of KN-62. The results showed no significant change in CAMK2 phosphorylation level, whereas the total CAMK2γ protein level was markedly reduced after KN-62 treatment (Figs. 2A–2C). Then, serum testosterone levels were assayed by the RIA and the results showed that KN-62 reduced the testosterone concentration by 66% compared with the control group (Fig. 2D). Additionally, the levels of 3β-HSD and StAR, two key steroidogenic enzymes, in testes were assayed via RT-qPCR and Western blot. The results showed that KN-62 treatment decreased Star and 3β-HSD mRNA levels by 54.5 and 77%, respectively (Figs. 2E, 2F), while protein levels were reduced by 62.6 and 58.5% (Figs. 2G–2I). These findings demonstrate that CAMK2 plays a critical role in testosterone production by regulating the expression of steroidogenic enzymes in mouse testes.

Fig. 2. Effects of CAMK2 on Testosterone Synthesis in Mouse Testes

(A) Western blot analysis and corresponding quantification (B, C) of phosphorylated CAMK2 and CAMK2γ in mouse testes with or without KN-62 treatment. (D) Serum testosterone levels in mice after 24-h KN-62 treatment. (E, F) RT-qPCR analysis of Star and 3β-HSD mRNA levels in mouse testes. (G–I) Western blot and analysis of StAR and 3β-HSD protein levels in mouse testes. Data are presented as mean ± S.E.M. (n ≥ 3). *p < 0.05; **p < 0.01; ***p < 0.001.

CAMK2 Has Positive Effect on Testosterone Synthesis in Cultured pLCs

To validate the in vivo findings, pLCs were isolated from PND 60 mice and identified via 3β-HSD immunofluorescence staining (Fig. 3A). The purity of the isolated pLCs was approximately 91.3% (Fig. 3B). Cultured pLCs were treated with KN-62 at the concentrations of 1, 10, 100 nM, 1, or 10 μM for 24 h. RIA measurements of testosterone levels in culture medium revealed that 1 and 10 μM KN-62 reduced testosterone levels by 59 and 52%, respectively, whereas lower concentrations (1, 10, 100 nM) had no significant effect (Fig. 3C). Consistent with the observed changes in testosterone levels, RT-qPCR and Western blot analysis demonstrated that 1 to 10 μM KN-62 significantly decreased both mRNA and protein expression of StAR and 3β-HSD (Figs. 3D–3H). These results indicate that CAMK2 positively regulates testosterone synthesis in pLCs, and its effect can be inhibited by 1 μM KN-62.

Fig. 3. Effects of KN-62 on Testosterone Synthesis in Mouse pLCs

(A) Isolated and identification of pLCs from PND 60 mouse testes. Immunofluorescence staining was performed using 3β-HSD (green) as a pLCs marker to assess cell purity. Nuclei were counterstained by DAPI (blue). Scale bars = 50 μm. (B) Proportion of 3β-HSD positive pLCs. ****p < 0.0001. (C) Testosterone levels in the culture medium of pLCs treated with increasing dose of KN-62 for 24 h. (D, E) RT-qPCR analysis of Star and 3β-HSD mRNA expressions in pLCs following KN-62 treatment. (F–H) Western blot and analysis of StAR and 3β-HSD protein in pLCs after 24 h of KN-62 treatment. Data are presented as mean ± S.E.M. Different superscript letters above the bars denote statistically significant differences (p < 0.05). pLCs: primary Leydig cells.

Inhibiting Effect of KN-62 on Testosterone Synthesis in Mouse pLCs Is Time Dependence

Since 1 μM KN-62 treatment for 24 h significantly inhibited testosterone production, we further examined the time-dependent effects of 1 μM KN-62 on pLCs over for 1, 3, 6, 12, and 24 h. Testosterone levels were reduced by 52 and 60.8% after 12 and 24 h KN-62 treatments, respectively, whereas 1, 3, and 6 h exposure had no significant effect compared with the control (Fig. 4A). RT-qPCR analysis showed that 12 and 24 h KN-62 treatment significantly decreased Star and 3β-HSD mRNA levels, while 1, 3, and 6 h treatments had no significant changes (Figs. 4B, 4C). Similarly, Western blot analysis demonstrated that StAR and 3β-HSD protein levels were significantly reduced only after 12 and 24 h of KN-62 exposure (by 56.9 and 72.6%, respectively), with no notable alterations at earlier time points (Figs. 4D–4F). We also examined the changes in CAMK2 activity in cultured pLCs after KN-62 treatment and found that KN-62 treatment did not significantly alter the phosphorylation level of CAMK2, but markedly reduced the CAMK2γ protein level (Figs. 4G–4I), which are consistent with the in vivo results (Figs. 2A–2C).

Fig. 4. Effects of KN-62 on Testosterone Synthesis Is Time-Dependence in pLCs

(A) Testosterone levels in the cultured medium following treatment with 1 μM KN-62 for 0, 1, 3, 6, 12, and 24 h. (B, C) RT-qPCR analysis of Star and 3β-HSD mRNA levels in pLCs treated with 1 μM KN-62 for indicated durations. (D–F) Western blot analysis of StAR and 3β-HSD protein in pLCs after KN-62 treatment for 0, 1, 3, 6, 12, and 24 h. (G) Western blot analysis and corresponding quantification (H, I) of phosphorylated CAMK2 and CAMK2γ in cultured pLCs with or without KN-62 treatment. Data are presented as mean ± S.E.M. (n = 3). *p < 0.05; **p < 0.01. pLCs: primary Leydig cells.

To further verify the function of CAMK2 in pLCs, we designed small interfering RNAs (siRNAs) targeting Camk2g and transfected them into pLCs, respectively, using Lipo8000. We then examined the knockdown efficiency of Camk2 and the resulting changes in testosterone synthesis. The results showed that all four synthesized siRNAs significantly reduced the mRNA level of Camk2g, with siCAMK2γ-1 and siCAMK2γ-2 showing the markedly inhibiting effects—57 and 60%, respectively (Fig. 5C). However, the four siRNAs also significantly decreased the level of Camk2b in pLCs (Fig. 5B), and siCAMK2γ-2 additionally reduced the Camk2d mRNA level (Fig. 5D). Although Camk2a mRNA levels were also decreased, the reductions were not significant (Fig. 5A), which may be related to the relatively high expression of Camk2b, Camk2g, Camk2d and very low expression of Camk2a in pLCs (Fig. 5E). Both siCAMK2γ-1 and siCAMK2γ-2 markedly inhibited the 3β-HSD and Star mRNA levels (Figs. 5G, 5H), as well as testosterone production in pLCs (Fig. 5F). These results indicate that siCAMK2γ we used not only interferes with Camk2g mRNA but also affects Camk2b and Camk2d mRNA levels, and that Camk2 knockdown suppresses testosterone synthesis in pLCs.

Fig. 5. Camk2 Knockdown Affects Testosterone Synthesis in pLCs

(A–D) RT-qPCR analysis of Camk2a, Camk2b, Camk2g and Camk2d mRNA in pLCs transfected with siCAMK2γ1-4 respectively. (E) RT-qPCR detection of Camk2a, Camk2b, Camk2g and Camk2d mRNA in pLCs. (F) Testosterone levels in the cultured medium transfected with siCAMK2γ-1 and siCAMK2γ-2. (G) 3β-HSD mRNA and (H)Star mRNA levels in pLCs transfected with siCAMK2γ-1 or siCAMK2γ-2 detected by RT-qPCR. Data are presented as mean ± S.E.M. (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001. pLCs: primary Leydig cells.

Collectively, these in vitro and in vivo results indicate that CAMK2 promotes testosterone synthesis, while KN-62 exerts inhibitory effects in a time- and dose-dependent manner. It is difficult to specifically knock down Camk2g by gene interference without affecting other isoforms in vitro.

CAMK2 Pathway Regulates Testosterone Synthesis

To determine whether CaMK2 functions as a downstream factor of known LH-regulated pathways, pLCs were, respectively, treated with inhibitors targeting key signaling molecules-10 μM MK-2206 (AKT inhibitor),³¹⁾ 100 nM CH (PKC inhibitor),³²⁾ 20 μM gefitinib (EGFR inhibitor),³³⁾ 25 μM H89 (PKA inhibitor),³⁴⁾ and 100 nM RAF709 (RAF1 inhibitor).³⁵⁾ None of these inhibitors significantly altered CAMK2γ expression (Figs. 6A, 6B), suggesting that CaMK2γ is not a downstream effector of the PKA or PKC pathways. Instead, these findings led us to propose that CaMK2 may act upstream of the EGFR/ERK1/2 pathway to regulate testosterone synthesis. To test this hypothesis, pLCs were treated with KN-62 prior to LH stimulation. Western blot analysis showed that KN-62 significantly reduced the phosphorylation of EGFR, ERK1/2, and the expression of RAF1 (Figs. 6C–6F). The in vivo results further demonstrated that inhibition of CAMK2 by KN-62 significantly reduced the levels of p-EGFR, RAF1, and p-ERK in the testis (Figs. 6G–6J). These data support the notion that CAMK2 may function upstream of the EGFR/RAF1/ERK1/2 pathway to modulate testosterone production. However, further studies are needed to fully elucidate the mechanistic interactions between CAMK2γ and this signaling cascade in testosterone synthesis.

Fig. 6. CAMK2 Is Involved in the EGFR/RAF1/ERK1/2 Signaling Pathway Regulating Testosterone Synthesis

(A, B) CAMK2γ protein levels in pLCs pretreated with inhibitors of AKT (MK-2206), PKC (CH), EGFR (gefitinib), PKA (H89), or RAF1 (RAF709). (C–F) Western blot analysis of phosphorylated (p) and total EGFR, ERK1/2, and RAF1 protein levels in pLCs treated with KN-62. (G–J) Western blot analysis of phosphorylated (p) and total EGFR, ERK1/2, and RAF1 protein levels in testis of mouse injected with or without KN-62. Data are presented as mean ± S.E.M. n = 3. *p < 0.05, **p < 0.01. pLCs: primary Leydig cells.

CAMK2 Is Involved in the Regulatory Effect of LH on Testosterone Synthesis by EGFR/RAF1/ERK Pathway

LH is a key regulator of testosterone synthesis, acting through multiple signaling pathways. To investigate whether CAMK2 is involved in the LH-mediated testosterone production, cultured pLCs were treated with 100 mIU/mL LH for 24 h. Western blot analysis revealed a significant increase in CAMK2γ protein (Figs. 7A, 7B), suggesting its potential role in LH signaling.

Fig. 7. CAMK2 Is Involved in the Regulatory Effect of LH on EGFR/RAF1/ERK Signaling Pathway in pLCs

(A, B) Detection of CAMK2γ protein in pLCs treated with 100 mIU/mL LH for 24 h. (C, D) RT-qPCR analysis of Star and 3β-HSD mRNA in pLCs treated with KN-62 for 12 h followed by 100 mIU/mL LH treatment for 24 h. (E) Western blot detection and (F–H) quantitative analysis of p-EGFR, EGFR, RAF1, p-ERK and ERK in pLCs treated with KN-62 for 12 h followed by 100 mIU/mL LH treatment for 24 h with GAPDH as internal control. Data are presented as mean ± S.E.M. n = 3. *p < 0.05, **p < 0.01, **p < 0.001. LH: luteinizing hormone; pLCs: primary Leydig cells.

To investigate whether CAMK2 is involved in LH-mediated regulation of testosterone synthesis, we pretreated cultured pLCs with 1 μM KN-62 for 12 h to inhibit CAMK2 activity, followed by treatment with 100 mIU/mL LH for 24 h. Subsequently, the expression changes of 3β-HSD and Star mRNA, as well as proteins in the EGFR/RAF1/ERK pathway, were examined. RT-qPCR results showed that KN-62 partially suppressed the LH-induced upregulation of 3β-HSD and Star mRNA in pLCs (Figs. 7C, 7D). Western blot analysis also indicated that KN-62 partially inhibited LH-induced phosphorylation of EGFR and ERK, as well as the upregulation of RAF1 protein (Figs. 7E–7H). These results indicate that CAMK2 contributes to the LH-mediated activation of the EGFR/RAF1/ERK pathway. However, after co-treatment with KN-62 and LH, both Star mRNA (Fig. 7D) and phosphorylated ERK levels (Fig. 7H) were still significantly elevated compared with the vehicle group, suggesting that CAMK2 participates in LH-stimulated testosterone synthesis, but blocking CAMK2 cannot completely abolish the promotive effect of LH on testosterone production.

DISCUSSION

This study demonstrates that CAMK2 plays a pivotal role in regulating testosterone synthesis in mouse Leydig cells. Our findings reveal that CAMK2 expression increases during testicular development, which correlates with the rise of testis testosterone content. Pharmacological inhibition of CAMK2 with KN-62 significantly suppressed testosterone production both in vivo and in vitro, accompanied by downregulation of key steroidogenic enzymes StAR and 3β-HSD. Furthermore, LH stimulation upregulated CAMK2 expression, suggesting its involvement in LH-mediated steroidogenesis. Importantly, we provide evidence that CAMK2 may act upstream of the EGFR/ERK1/2 signaling cascade to regulate testosterone synthesis. These findings highlight CAMK2 as a novel regulator of Leydig cell function and a potential therapeutic target for male reproductive disorders.

Our study is the first to characterize the developmental expression pattern of CAMK2 isoforms in mouse testes. We found that Camk2d was the most abundant isoform, while Camk2g exhibited a significant increase post-puberty, coinciding with the onset of active testosterone production.⁶⁾ Since testosterone synthesis is low during infancy but surges after puberty,^6,9) the upregulation of Camk2g in adulthood suggests its critical role in supporting steroidogenesis. This temporal expression pattern aligns with the functional maturation of Leydig cells,^36,37) which acquire full steroidogenic capacity during postnatal development. Immunohistochemical analysis confirmed that CAMK2γ is predominantly localized in the cytoplasm of Leydig cells, consistent with its involvement in intracellular signaling pathways.^22,24) Additionally, CAMK2 is a calcium-sensitive kinase, our findings align with previous studies demonstrating that calcium signaling is essential for steroidogenesis.³⁸⁾ This is further supported by the fact that cholesterol transport and steroidogenic enzyme activity are calcium-dependent processes.

The most compelling evidence for CAMK2’s role in testosterone synthesis comes from experiments using its pharmacological inhibitor KN-62. KN-62 was originally published as a specific inhibitor of CAMK2 by Hiroshi Tolumitsu and his colleagues.²⁹⁾ It was also reported to have minimal effects on other kinases but has similar inhibitory potency on CAMK1 and CAMK4;^39,40) meanwhile, CAMK1 was demonstrated to expressed in Leydig cells.⁴¹⁾ In addition to using KN-62, we also designed siRNAs targeting Camk2g to investigate its function in pLCs. However, due to the high sequence similarity among different Camk2 isoforms, these siRNAs could not specifically target Camk2g; the siRNAs also affected the expression of Camk2b mRNA, Camk2g mRNA, or Camk2d mRNA. Therefore, we relied on the more specific pharmacological tool (KN-62) to delineate the signaling pathway. Future studies using isoform-specific genetic models are needed to definitively map the signaling cascade. Nevertheless, our siRNA interference experiments demonstrated that knockdown of Camk2 inhibited testosterone synthesis in Leydig cells.

In vivo inhibition led to a significant reduction in serum testosterone levels, accompanied by downregulation of key steroidogenic enzymes, including StAR and 3β-HSD, all of which are critical for cholesterol-to-testosterone conversion.^6,42) In vitro studies in primary Leydig cells further confirmed that KN-62 treatment suppressed steroidogenic enzyme expression and testosterone production in a dose- and time-dependent manner, suggesting that CAMK2 regulates these enzymes at the transcriptional or post-transcriptional level. The delayed suppression of KN-62 on StAR and 3β-HSD expression may be attributed to the time required for KN-62 to effectively reduce CAMK2 protein level. Unlike the rapid activation of steroidogenic genes by LH or hCG, which typically occurs within 0.5–6 h through cAMP/PKA-dependent signaling,^43,44) KN-62 acts as an upstream kinase inhibitor and may exert its effects by inhibiting CAMK2 protein level in our research. This differential effect may be interpreted through the distinct mechanisms of KN-62 action and CaMKII regulation. KN-62 acts as a Ca²⁺/calmodulin (CaM) antagonist, preventing new CaMK2 activation events but not reversing pre-existing Thr286/287 autophosphorylation. Once CaMK2γ becomes autophosphorylated, the phosphorylated state is CaM-independent and relative stable.²⁹⁾ By contrast, the reduction in CaMK2γ protein levels likely reflects a slower regulation at the transcriptional or proteostatic level, leading to different responses between autophosphorylation and total protein abundance. However, while KN-62 treatment markedly reduced testosterone synthesis, the effect was incomplete, implying that compensatory pathways may partially sustain steroidogenesis in the absence of CAMK2 activity. This observation, along with previous studies,^42,45) suggests that although CAMK2 is a key regulator, other signaling mechanisms may also contribute to testosterone production, warranting further investigation.

Since LH is the primary regulator of testosterone synthesis, we investigated whether CAMK2 mediates LH’s steroidogenic effects. Our data show that LH upregulates CAMK2γ expression, positioning it as a potential mediator of LH signaling. LH classically acts through PKA, PKC, and MAPK pathways,^4,46–49) which lead to the upregulation of steroidogenic enzymes and subsequent testosterone production. However, our inhibitor studies revealed that CAMK2 is not a downstream effector of PKA, PKC, AKT or RAF1. Instead, we found that CAMK2 inhibition suppressed EGFR and ERK1/2 phosphorylation and weakened the promoting effect of LH on testosterone production, suggesting that it may function upstream of the EGFR/RAF1/ERK1/2 pathway, a well-established modulator of LH-induced steroidogenesis.⁴⁶⁾ RAF1 was demonstrated to be phosphorylated at Ser338 (S338), a modification that depends on RAS signaling activated by epidermal growth factor (EGF) and its receptor EGFR.⁵⁰⁾ These phosphorylation events induce conformational changes that relieve RAF1 autoinhibition, enabling its kinase domain to become catalytically active and subsequently transmit signals to the downstream MEK-ERK1/2 pathway.⁵¹⁾ Consistent with our results that KN-62 inhibited RAF1 protein levels in testis and pLCs, another study reported that EGF-EGFR signaling can activate the RAS-RAF-MEK-ERK1/2 pathway by upregulating the expression of RAS and RAF1 proteins.⁵²⁾ Recent research reveals that the C-terminal to LisH (CTLH) complex, a multiprotein E3 ubiquitin ligase complex, promotes RAF1 ubiquitination and degradation, thereby controlling RAF1 protein level.⁵³⁾ Based on this evidence, it is reasonable to hypothesize that EGF-EGFR signaling may inhibit CTLH-mediated ubiquitination of RAF1, preventing its degradation and leading to RAF1 accumulation.

Multiple studies have shown that CaMK2 acts upstream of the EGFR signaling pathway by phosphorylating and promoting the activation of EGFR.^54,55) The activation of EGFR subsequently regulates RAF1 expression and ERK1/2 phosphorylation.^52,56–58) In our present study, one plausible mechanism is that CAMK2 phosphorylates and activates EGFR,⁵⁹⁾ thereby enhancing ERK1/2 signaling and subsequent steroidogenic gene expression. However, the precise molecular interactions remain unclear and merit further investigation. Co-immunoprecipitation assays could determine whether CAMK2 physically associates with EGFR or intermediate signaling molecules, while genetic approaches (knockout mouse model) could provide definitive evidence of its role in steroidogenesis.

In summary, our study establishes CAMK2 as a key regulator of testosterone biosynthesis in Leydig cells, acting through the EGFR/ERK1/2 pathway. These findings expand our understanding of the molecular mechanisms controlling steroidogenesis and highlight CAMK2 as a potential therapeutic target for male reproductive disorders. Future research is needed to fully dissect its signaling interactions and assess its clinical applicability.

Acknowledgments

This work was supported by the Natural Science Foundation of Jiangsu Province (Grant No. BK20230581), the Jiangsu Entrepreneurship and Innovation Teams Project (JSSCTD202224), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

DECLARATIONS

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

All data generated or analyzed during this study are available from the corresponding author on reasonable request.

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