GJB2 drives LUAD progression in part via cAMP-mediated M2 macrophage polarization through the PKA-CREB pathway

Abstract

Background

M2 macrophage polarization is a key driver of lung adenocarcinoma (LUAD) progression, yet its underlying regulatory mechanisms remain unclear. The expression pattern and biological function of the gap junction protein GJB2 in LUAD have not been elucidated to date.

Methods

TCGA database analysis was used to determine GJB2 expression in LUAD and its correlation with prognosis. GJB2 knockdown (KD) and overexpression (OE) models were established in A549/NCI-H1975 cells. EdU, Transwell, and flow cytometry assays evaluated LUAD cell proliferation, migration, invasion, and apoptosis. A LUAD-THP-1 macrophage co-culture system, cAMP agonist/inhibitor treatment, and PKA-CREB pathway analysis were applied to explore mechanisms. Nude mouse xenograft models validated findings in vivo.

Results

GJB2 was significantly upregulated in LUAD tissues and correlated with reduced overall survival. GJB2-KD inhibited LUAD cell malignant behaviors and promoted apoptosis, while GJB2-OE exerted opposite effects. GJB2 mediated gap junction-dependent cAMP transfer to macrophages, activating the PKA-CREB axis to induce M2 polarization; cAMP agonists reversed GJB2-KD effects. In vivo, GJB2-KD suppressed tumor growth, reduced serum cAMP and M2 cytokines, and inhibited PKA/CREB phosphorylation.

Conclusions

The GJB2-cAMP-PKA-CREB axis drives LUAD progression by inducing M2 macrophage polarization. GJB2 may serve as a novel prognostic biomarker and potential therapeutic target for LUAD.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12931-026-03537-1.

Introduction

Lung cancer is the malignant tumor with the highest incidence and mortality worldwide, imposing an increasingly heavy disease burden. Among its subtypes [1, 2], lung adenocarcinoma (LUAD) accounts for over 40% of cases, making it the most common pathological subtype. Although targeted therapy and immunotherapy have improved the prognosis of some patients, LUAD exhibits insidious early symptoms, and most patients are diagnosed at an advanced stage, resulting in a still low 5-year survival rate [3]. Therefore, clarifying the molecular mechanisms underlying the occurrence and development of LUAD and identifying novel therapeutic targets are of great significance for improving patient prognosis.

In the tumor microenvironment (TME), immune cells exert both anti-tumor and pro-tumor effects. As the most abundant immune cells in the TME, macrophages possess high plasticity and can differentiate into two phenotypes: M1 (pro-inflammatory, anti-tumor) and M2 (anti-inflammatory, pro-tumor). M1 macrophages secrete cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) to enhance anti-tumor immunity, while M2 macrophages secrete factors including interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) to promote tumor growth and metastasis [4]. Studies have confirmed that the infiltration of M2 macrophages is increased in LUAD and correlates with poor prognosis [5]. Regulating macrophage polarization may thus represent a therapeutic strategy for LUAD; however, the specific mechanism by which LUAD cells regulate macrophage polarization remains unclear.

Gap junctions (GJs) are composed of connexins (Cxs) and mediate the transfer of small molecules between cells, participating in various physiological and pathological processes. The GJB2 gene encodes connexin 26 (Cx26), a core protein of GJs. Initially identified to be associated with hereditary deafness, recent studies have shown that GJB2 is abnormally expressed in tumors such as breast cancer and cervical cancer, and its expression correlates with tumor biological behavior and prognosis [6, 7]. However, the expression and function of GJB2 in LUAD, as well as its potential involvement in regulating macrophage polarization, have not been reported to date.

Cyclic adenosine monophosphate (cAMP), a second messenger, regulates macrophage polarization by activating the PKA-CREB pathway: increased cAMP levels activate PKA, which phosphorylates CREB; phosphorylated CREB (p-CREB) then translocates to the nucleus, binds to specific DNA sequences, and regulates the expression of target genes to promote M2 polarization [8]. Nevertheless, it remains unclear whether GJB2 mediates cAMP transfer between tumor cells and macrophages to regulate the PKA-CREB pathway and macrophage polarization in LUAD.

Based on this background, the present study investigated the role and mechanism of GJB2 in LUAD: TCGA database analysis was used to determine the expression of GJB2 in LUAD and its correlation with prognosis; GJB2 knockdown/overexpression (GJB2-KD/OE) models were established in A549 and NCI-H1975 cells to examine the effect of GJB2 on the biological behavior of LUAD cells; a co-culture system of LUAD cells and THP-1-derived macrophages was constructed to explore the regulatory role of GJB2 in macrophage polarization; the role of cAMP as an intercellular communication molecule and the mediating effect of the PKA-CREB pathway were investigated; and in vivo validation was conducted using a nude mouse xenograft model. This study aims to reveal a novel mechanism of GJB2 in LUAD and provide a potential target for LUAD therapy.

Materials and methods

Cell culture

Human LUAD cell lines A549/NCI-H1975 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and monocytic THP-1 (ATCC) were used. A549/NCI-H1975 were cultured in RPMI-1640 medium with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco); THP-1 in RPMI-1640 with 10% FBS. All cells were maintained at 37 °C, 5% CO₂ in a humidified incubator, with medium refreshed every 2–3 days and passaged/treatment at 80%–90% confluence.

GJB2 knockdown (KD) and overexpression (OE)

GJB2-targeting shRNA was cloned into lentiviral vector pLKO.1 (Addgene). Lipofectamine 3000 co-transfected this vector with packaging/envelope plasmids into 293T cells to produce lentivirus. Viral supernatant infected A549/NCI-H1975, with positive cells selected by 2 µg/mL puromycin; KD efficiency was verified by WB.

Full-length GJB2 recombinant plasmid was transfected into A549/NCI-H1975. Stable OE cell lines were obtained via antibiotic selection, with efficiency confirmed by WB.

WB analysis

Cells were washed with PBS, lysed on ice with RIPA buffer for 30 min, centrifuged (12,000×g, 15 min) to collect supernatant. Protein concentration was measured by bicinchoninic acid assay. Equal proteins (mixed with 5×SDS-PAGE loading buffer, boiled 5 min) were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk (1 h, RT), then incubated overnight (4℃) with primary antibodies: GJB2 (ab303498, Abcam), CREB (12208-1-AP, Proteintech), p-CREB (81871-1-RR, Proteintech), Arg1 (16001-1-AP, Proteintech), IL10 (82191-3-RR, Proteintech), β-actin (20536-1-AP, Proteintech) (all 1:500). Next day, membranes were incubated with HRP-conjugated secondary antibody (SA00001-2, Proteintech, 1:2000) for 1 h (37℃). Bands were visualized by ECL, grayscale analyzed via ImageJ.

Cell proliferation assay

Transfected A549/NCI-H1975 (1 × 10⁶ cells/well, 6-well plate) were cultured 24 h, then incubated with 10 µmol/L EdU (C00053, Ribobio) for 2 h. Cells were washed with PBS, digested with 0.25% trypsin, terminated with 2% FBS-PBS, centrifuged (300×g, 5 min), fixed with 4% PFA (4℃, 30 min), permeabilized with 0.3% Triton X-100 (15 min, RT). After 30 min dark incubation with 500 µL Click solution, cells were washed with PBS. EdU fluorescence was detected by flow cytometer (488 nm laser, FITC channel), PI excluded dimers. ≥2 × 10⁴ cells/sample were collected; proliferation index was calculated via FlowJo v10.

Transwell assays

Uncoated chambers (migration) and Matrigel-coated chambers (invasion) were used. Transfected cells (serum-free medium) were seeded into upper chambers; lower chambers contained 10% FBS-medium. After 24 h culture, cells were fixed with 4% PFA, stained with 0.1% crystal violet, and counted under light microscope.

Flow cytometry

Apoptosis was detected via CoraLite^® Plus 488-Annexin V/PI Kit (PF00005, Proteintech). Macrophage markers were labeled with iNOS-CoraLite^® Plus 647 (18985-1-AP, Proteintech), CD86-CoraLite^® Plus 488 (65165-1-Ig, Proteintech), CD206-CoraLite^® Plus 488 (98031-1-RR, Proteintech), CD163-CoraLite^® Plus 647 (65561-1-MR, Proteintech). After PBS washing, flow cytometry determined M1/M2 proportion.

GJB2/PKA detection

Mouse xenograft tumor paraffin sections were dewaxed; co-cultured cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, blocked with 5% BSA. Samples were incubated overnight (4℃) with fluorescent primary antibodies: GJB2 (ab303498, Abcam), PKA (27398-1-AP, Proteintech). Next day, samples were incubated with FITC-conjugated goat anti-rabbit IgG (SA00003-2, Proteintech) and DAPI (PR30021, Proteintech) for nuclear staining. Mounted with anti-fluorescence quenching medium, signals were captured via microscope.

cAMP detection

cAMP levels were measured via ELISA Kit (E-EL-0056, Elabscience). Transfected LUAD cells/supernatants were collected: cells washed 2–3× with PBS, lysed on ice (15–30 min, RIPA with protease/phosphatase inhibitors), centrifuged (12,000 rpm, 15 min) to collect supernatant. Concentration (pmol/mL) was determined per kit instructions; experiments repeated 3×.

cAMP Agonist/Inhibitor treatment

GJB2-KD group was treated with 100 µmol/L 8-Br-cAMP (B7880, Sigma); GJB2-OE group with 10 µmol/L H-89 (371962, Sigma) for 24 h; blank control was set. Macrophage polarization was detected by flow cytometry, related proteins by WB.

Cytokine ELISA

Cell supernatants were collected; IL-10 (MU30055, HM10203, Bioswamp), TGF-β (MU30071, HM10058, Bioswamp), IL-4 (MU30385, HM10380, Bioswamp) levels were measured via ELISA kits per instructions; results expressed as pg/mL.

Animal experiments

SPF-grade BALB/c nude mice (female, 4–6 weeks old, Beijing Weitong Lihua) were housed in SPF facility. A549/NCI-H1975 were divided into NC, GJB2-KD, GJB2-OE groups (6 mice/group). 1 × 10⁶ cells/mouse were subcutaneously inoculated into dorsal region. Tumor volume (length×width²×0.5) was measured every 3 days. On day 21, mice were euthanized; tumors were excised/weighed. Serum detected cAMP/cytokines; tumor tissues analyzed by WB/immunofluorescence.

Statistical analysis

Experiments repeated ≥ 3×; data expressed as mean ± SD (x ± s). Analyzed via GraphPad Prism 9.5: independent samples t-test (two groups), one-way ANOVA + LSD-t test (multiple groups). P < 0.05 was statistically significant.

Results

GJB2 upregulation in LUAD correlates with poor prognosis

To clarify the expression characteristics and clinical significance of GJB2 in LUAD, we first analyzed GJB2 expression in LUAD patients using data from The Cancer Genome Atlas (TCGA) database. Results showed that GJB2 expression was significantly higher in LUAD tumor tissues than in adjacent normal tissues (Fig. 1A). Further survival analysis revealed a significant negative correlation between GJB2 expression and overall survival (OS) of LUAD patients (Fig. 1B), with patients with high GJB2 expression exhibiting markedly shorter OS.

Fig. 1.

Upregulation of GJB2 in LUAD Correlates with Survival and Prognosis. A-B The expression level of GJB2 in LUAD tumor tissues (A) and its impact on the survival of LUAD patients (B) were analyzed using the TCGA database. C-F WB was performed to verify the effects of GJB2-KD and GJB2-OE in A549 (C-D) and NCI-H1975 (E-F) cells. G-H Flow cytometry was used to determine the effect of GJB2-KD/OE on the proliferation ability of A549 (G) and NCI-H1975 (H) cells. I-J Transwell assay was conducted to assess the effect of GJB2-KD/OE on the migration and invasion abilities of A549 (I) and NCI-H1975 (J) cells. K-L Flow cytometry was employed to measure the effect of GJB2-KD/OE on the apoptosis rate of A549 (K) and NCI-H1975 (L) cells. All data are presented as means ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test and the student’s t test. Statistically significant differences were indicated by *p < 0.05 and **p < 0.01

To verify the effect of GJB2 on the biological behavior of LUAD cells, GJB2-KD and GJB2-OE models were established in the LUAD cell lines A549 and NCI-H1975 (Figs. 1C-F). Functional assays were then performed to evaluate changes in cell proliferation, migration, invasion, and apoptosis. Cell proliferation assays demonstrated that compared with the NC group, GJB2-KD significantly inhibited the proliferative capacity of A549 and NCI-H1975 cells, whereas GJB2-OE significantly enhanced cell proliferation (Figs. 1G-H). Transwell assays showed that GJB2-KD markedly suppressed the migration and invasion of LUAD cells, while GJB2-OE exerted the opposite effect, significantly promoting cell migration and invasion (Figs. 1I-J). Flow cytometry analysis of apoptosis indicated that GJB2-KD significantly increased the apoptotic rate of LUAD cells, whereas GJB2-OE significantly reduced apoptosis levels (Figs. 1K-L). Collectively, these results confirm that high GJB2 expression promotes LUAD progression by enhancing LUAD cell proliferation, migration, and invasion, while regulating cell apoptosis.

GJB2 promotes LUAD progression by inducing M2 macrophage polarization

To explore the potential mechanism by which GJB2 regulates LUAD progression, we first analyzed the correlation between GJB2 expression and immune cell infiltration in the LUAD TME using TCGA data. Results showed a significant positive correlation between GJB2 expression and macrophage infiltration (Fig. 2A), and previous studies have confirmed that macrophage polarization status is closely associated with tumor progression [9].

Fig. 2.

GJB2 Promotes LUAD Progression by Inducing M2 Macrophage Polarization. A The correlation between GJB2 expression and macrophage infiltration in the LUAD tumor microenvironment was analyzed via the TCGA database. B-C Confocal microscopy was used to observe the subcellular localization of GJB2 in co-culture systems of A549 (B) / NCI-H1975 (C) cells and THP-1-derived macrophages. D-E Flow cytometry was performed to determine the proportions of M1 and M2 macrophages in co-culture systems of A549 (D) / NCI-H1975 (E) cells and THP-1 cells after GJB2-KD/OE. F-G Flow cytometry was used to measure the proliferation ability of GJB2-KD/OE A549 (F) and NCI-H1975 (G) cells following co-culture with M2-type macrophages. H-I Transwell assay was conducted to evaluate the migration and invasion abilities of GJB2-KD/OE A549 (H) and NCI-H1975 (I) cells after co-culture with M2-type macrophages. J-K Flow cytometry was employed to assess the apoptosis ability of GJB2-KD/OE A549 (J) and NCI-H1975 (K) cells following co-culture with M2-type macrophages. All data are presented as means ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test and the student’s t test. Statistically significant differences were indicated by *p < 0.05 and **p < 0.01

To clarify the localization of GJB2 between LUAD cells and macrophages, the LUAD cell lines (A549, NCI-H1975) were co-cultured with THP-1-derived macrophages, and the subcellular localization of GJB2 was detected by laser confocal microscopy. In both co-culture systems, GJB2 was localized to the cell membranes of both LUAD cells and THP-1 cells, with the formation of intercellular junction structures between the two cell types (Figs. 2B-C). This result is consistent with the biological function of GJB2 as a core protein of gap junction channels.

To further investigate the effect of GJB2 on macrophage polarization, A549 and NCI-H1975 cells with GJB2-KD or OE were co-cultured with THP-1 cells, and the expression levels of M1 (CD86, iNOS) and M2 (CD206, CD163) macrophage markers were detected. Results showed that GJB2-KD significantly increased the proportion of M1 macrophages while decreasing the proportion of M2 macrophages; in contrast, GJB2-OE showed the opposite trend, reducing the proportion of M1 macrophages and increasing the proportion of M2 macrophages (Figs. 2D-E). These findings suggest that GJB2 can promote the polarization of macrophages toward the M2 phenotype.

To verify the effect of M2 macrophages on the biological behavior of LUAD cells, LUAD cells with GJB2-KD/OE were co-cultured with M2 macrophages, and cell proliferation, migration, invasion, and apoptosis were detected. Regardless of GJB2 expression status in LUAD cells, M2 macrophages significantly promoted LUAD cell proliferation (Figs. 2F-G) and migration/invasion (Figs. 2H-I), while significantly inhibiting LUAD cell apoptosis (Figs. 2J-K). Taken together, these results indicate that GJB2 accelerates LUAD progression by promoting M2 macrophage polarization.

GJB2 induces M2 polarization by mediating cAMP transfer between LUAD cells and THP-1 cells

Based on previous findings that GJB2 mediates small molecule transfer via gap junctions [10], we hypothesized cAMP is a key regulator. GJB2-KD reduced cAMP levels in LUAD cell supernatants, while GJB2-OE increased them (Figs. 3A-B). Supernatant co-culture assays showed GJB2-KD supernatant promoted M1 polarization, and GJB2-OE supernatant promoted M2 polarization (Figs. 3C-D). Adding a cAMP agonist to GJB2-KD supernatants reversed M1 polarization and enhanced M2 polarization, while a cAMP inhibitor had the opposite effect on GJB2-OE supernatants (Figs. 3E-H), confirming GJB2 induces M2 polarization via cAMP transfer.

Fig. 3.

GJB2 Induces M2 Polarization by Mediating cAMP Transfer Between LUAD Cells and THP-1 Cells. A-B ELISA was used to detect the relative ratio of cAMP in the supernatant of GJB2-KD/OE A549 (A) and NCI-H1975 (B) cells. C-D Flow cytometry was performed to determine the polarization status of THP-1 cells after co-culture with GJB2-KD/OE A549 (C) and NCI-H1975 (D) cells. E-F Flow cytometry was used to measure the polarization status of THP-1 cells following co-culture with GJB2-KD A549 (E) and NCI-H1975 (F) cells supplemented with a cAMP activator. G-H Flow cytometry was employed to assess the polarization status of THP-1 cells after co-culture with GJB2-OE A549 (G) and NCI-H1975 (H) cells supplemented with a cAMP inhibitor. All data are presented as means ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test and the student’s t test. Statistically significant differences were indicated by *p < 0.05 and **p < 0.01

cAMP regulates THP-1 cell polarization via the PKA-CREB pathway

Previous studies have shown that cAMP, as a second messenger, binds to the regulatory subunit of PKA to activate PKA; activated PKA then phosphorylates the downstream target protein CREB. P-CREB translocates to the nucleus, binds to specific DNA sequences, and thereby regulates macrophage polarization [8]. Based on this, LUAD cells with GJB2-KD/OE were co-cultured with THP-1 cells, and a cAMP agonist was added to the GJB2-KD group while a cAMP inhibitor was added to the GJB2-OE group. WB was used to detect the protein expression levels of CREB, p-CREB, and M2 macrophage markers (Arg-1, IL-10) in THP-1 cells. Compared with the A549/NCI-H1975-GJB2-KD + M0 group, treatment with the cAMP agonist significantly upregulated the protein expression levels of p-CREB, Arg-1, and IL-10 (Figs. 4A-B); in contrast, compared with the A549/NCI-H1975-GJB2-OE + M0 group, treatment with the cAMP inhibitor significantly downregulated the expression of these proteins (Figs. 4A-B).

Fig. 4.

cAMP Regulates THP-1 Cell Polarization via the PKA-CREB Pathway. A-B Western blot was performed to determine the protein expression levels of PKA, CREB, p-CREB, Arg-1, and IL-10 in THP-1 cells co-cultured with A549 (A) and NCI-H1975 (B) cells. C-D ELISA was used to detect the secretion levels of IL-10, TGF-β, and IL-4 in THP-1 cells co-cultured with A549 (C) and NCI-H1975 (D) cells. E-F: Confocal microscopy was employed to observe the subcellular localization of PKA in THP-1 cells co-cultured with A549 (E) and NCI-H1975 (F) cells. All data are presented as means ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test and the student’s t test. Statistically significant differences were indicated by *p < 0.05 and **p < 0.01

Meanwhile, ELISA was used to detect the secretion levels of IL-10, TGF-β, and IL-4 by THP-1 cells. Results showed that treatment with the cAMP agonist significantly increased the secretion of these cytokines (Figs. 4C-D), while treatment with the cAMP inhibitor significantly reduced their secretion (Figs. 4C-D). In addition, IF showed that treatment with the cAMP agonist significantly promoted the nuclear translocation of PKA in THP-1 cells, whereas treatment with the cAMP inhibitor significantly inhibited PKA nuclear translocation (Figs. 4E-F). Collectively, these results confirm that cAMP regulates the polarization of THP-1 cells toward the M2 phenotype by activating the PKA-CREB signaling pathway.

In vivo validation via animal experiments

To verify the reliability of the aforementioned mechanism in vivo, a subcutaneous xenograft model was established in nude mice, which were inoculated with LUAD-NC, LUAD-GJB2-KD, or LUAD-GJB2-OE cells. Nude mouse xenograft models showed GJB2-KD inhibited tumor growth (reduced volume and weight), while GJB2-OE promoted it (Figs. 5A-B). Serum ELISA detected decreased cAMP and M2 cytokine levels in the GJB2-KD group and increased levels in the GJB2-OE group (Figs. 5C-F). Flow cytometry, WB, and IF confirmed GJB2-KD promoted M1 polarization, inhibited M2 polarization, and suppressed the PKA-CREB pathway, whereas GJB2-OE activated this axis (Figs. 5G-L). In vivo data further confirm GJB2 mediates cAMP transfer, activates PKA-CREB signaling, induces M2 polarization, and drives LUAD progression.

Fig. 5.

In Vivo Validation via Animal Experiments. A-B The effects of inoculating GJB2-KD/OE A549 (A) and NCI-H1975 (B) cells on tumor volume and weight were analyzed in a nude mouse subcutaneous tumor model. C-F ELISA was used to detect the relative ratio of cAMP and the levels of IL-10, TGF-β, and IL-4 in the serum of nude mice inoculated with GJB2-KD/OE A549 (C&E) and NCI-H1975 (D&F) cells. G-H Flow cytometry was performed to determine the M1/M2 polarization status of macrophages in tumor tissues of nude mice inoculated with GJB2-KD/OE A549 (G) and NCI-H1975 (H) cells. I-J WB was used to measure the protein expression levels of PKA, CREB, and p-CREB in tumor tissues of nude mice inoculated with GJB2-KD/OE A549 (I) and NCI-H1975 (J) cells. K-L IF was employed to observe the expression and nuclear translocation of PKA in tumor tissues of nude mice inoculated with GJB2-KD/OE A549 (K) and NCI-H1975 (L) cells. All data are presented as means ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test and the student’s t test. Statistically significant differences were indicated by *p < 0.05 and **p < 0.01

In summary, in vivo experiments further confirm that GJB2 mediates cAMP transfer via gap junctions, activates the PKA-CREB signaling axis, induces M2 macrophage polarization, and thereby promotes LUAD progression.

Discussion

LUAD, the most prevalent lung cancer subtype, has high mortality due to late diagnosis and limited therapeutic targets. Elucidating its molecular mechanisms is critical for improving prognosis. This study is the first to demonstrate that gap junction protein GJB2 promotes LUAD progression by mediating cAMP transfer between LUAD cells and macrophages, activating the PKA-CREB pathway to induce M2 polarization—providing a novel theoretical basis and potential therapeutic target for LUAD.

TCGA analysis showed GJB2 was significantly upregulated in LUAD tissues, with high expression negatively correlated with patients’ overall survival, suggesting GJB2 as a poor-prognosis biomarker. This aligns with GJB2’s pro-tumor role in other cancers: high GJB2 expression reduces 5-year disease-free survival in triple-negative breast cancer [7], and drives hepatocellular carcinoma (HCC) progression by regulating glycolysis and the tumor microenvironment (TME) [11]. Our study is the first to confirm GJB2’s high expression and pro-tumor effect in LUAD, though further experiments are needed to validate its specific mechanisms.

In vitro assays revealed GJB2-KD inhibited proliferation, migration, and invasion of A549/NCI-H1975 cells while increasing apoptosis, with GJB2-OE exerting opposite effects. Mechanistically, GJB2 exerts its cell-autonomous regulation of LUAD cell proliferation, migration, invasion, and apoptosis by modulating key signaling cascades and effector molecules. GJB2 overexpression can enhance the activity of pro-proliferative pathways (e.g., MAPK/ERK and PI3K/AKT) to upregulate cyclin D1 expression and accelerate cell cycle progression from G1 to S phase. For migration and invasion, GJB2 promotes the EMT process by upregulating the expression of EMT transcription factors and matrix metalloproteinases, thereby enhancing the extracellular matrix degradation capacity and cell motility of LUAD cells. In terms of apoptosis regulation, GJB2 overexpression downregulates the expression of pro-apoptotic proteins (e.g., Bax, Caspase-3) and upregulates anti-apoptotic proteins (e.g., Bcl-2, Bcl-xL), leading to the suppression of mitochondrial apoptosis pathway activation. Conversely, GJB2 knockdown abrogates these effects, blocking cell cycle progression, reversing EMT, and promoting apoptotic cell death in LUAD cell lines. GJB2’s role in LUAD shares similarities (driving tumor progression via enhancing malignant behaviors) and differences (apoptosis regulation) with other cancers: pan-cancer studies show GJB2 inhibits apoptosis via PI3K/AKT [12], but we found GJB2-OE reduced LUAD cell apoptosis. This discrepancy may stem from GJB2 directly regulating apoptosis-related proteins (e.g., Bcl-2, Caspase-3) or indirectly via TME macrophages—supported by co-culture experiments showing M2 macrophages inhibit LUAD apoptosis, suggesting “cell-autonomous” and “TME-dependent” regulatory modes. Further studies (cell sorting, single-cell RNA sequencing) are needed to explore this.

Dysregulated macrophage polarization in the TME drives LUAD progression; M2 infiltration in LUAD tissues promotes tumorigenesis via IL-10/TGF-β [13], but how LUAD cells regulate this remains unclear. Our TCGA analysis showed GJB2 expression positively correlated with macrophage infiltration, and co-culture experiments confirmed GJB2 promotes M2 polarization of THP-1-derived macrophages, which in turn enhance LUAD cell malignancy—forming a “high GJB2-LUAD cells-M2 macrophages-LUAD progression” positive feedback loop, offering new insights into TME regulation.

Laser confocal microscopy showed GJB2 localizes to LUAD cell and macrophage membranes, forming intercellular junctions—consistent with its gap junction function. Gap junctions in TME communication are increasingly studied: GJB2 mediates tumor-stellate cell communication to promote fibrosis [14], and GJB4 drives gastric cancer via Wnt/β-catenin [15]. Our study is the first to link GJB2-mediated gap junctions to LUAD macrophage polarization, expanding understanding of gap junctions in the tumor immune microenvironment.

Compared with other mechanisms that regulate macrophage polarization, GJB2 is unique in its “direct communication” mode. Previous studies have shown that LUAD cells induce M2 polarization via “paracrine” regulation, such as secreting exosomes to deliver miR-19b-3p [16] or releasing IL-6 to activate the STAT3 pathway [17]. In contrast, GJB2 directly transfers signaling molecules via gap junctions—a process that is faster and more efficient, suggesting it may play a critical role in the early stages of LUAD progression. Additionally, in the Transwell non-contact co-culture system, GJB2 still modulated macrophage polarization by regulating soluble factors secreted by LUAD cells—indicating that GJB2 may regulate polarization through both “direct gap junction communication” and “indirect paracrine communication.” This provides a comprehensive framework for understanding the role of GJB2 in the LUAD TME.

Identifying the downstream signaling molecules through which GJB2 regulates macrophage polarization is key to elucidating its mechanism. Based on the ability of gap junctions to transfer small molecules (e.g., cAMP) [18], we hypothesized that cAMP is a key mediator. Experiments confirmed that GJB2 knockdown reduced cAMP levels in LUAD cell supernatants, whereas GJB2 overexpression increased these levels. Furthermore, cAMP in LUAD cell supernatants directly regulated macrophage polarization: adding a cAMP agonist reversed the inhibition of M2 polarization induced by GJB2 knockdown, while adding a cAMP inhibitor blocked the promotion of M2 polarization by GJB2 overexpression. This is the first study to confirm that cAMP is a key signaling molecule mediating communication between LUAD cells and macrophages via GJB2.

The role of cAMP in macrophage polarization has been widely reported: drugs that increase cAMP levels (e.g., forskolin, db-cAMP) induce the M2 phenotype and inhibit M1 marker expression in both mouse and human macrophages [19]; activation of the cAMP-PKA pathway in human monocyte-derived macrophages upregulates M2 markers such as CD206 and arginase-1 (Arg-1) [20]. Our study further confirmed that the role of cAMP in LUAD macrophage polarization is dependent on the PKA-CREB pathway: GJB2 overexpression upregulated CREB phosphorylation, and PKA nuclear translocation in macrophages, and these effects were reversed by adding a cAMP inhibitor. The expression of M2 markers (Arg-1, IL-10) was consistent with PKA-CREB pathway activity, and ELISA results showed that the cAMP agonist increased the secretion of IL-10, TGF-β, and IL-4. These findings align with previous studies and establish a complete molecular cascade: “GJB2 - gap junctions - cAMP-PKA-CREB - M2 polarization.”

It should be noted that cAMP may also regulate macrophage polarization via other pathways. Previous studies have confirmed that cAMP can activate the Epac pathway, and Epac activation promotes M2 polarization in macrophages [21]; cAMP may also affect macrophage phenotypes by regulating histone deacetylases [22]. In our study, the PKA inhibitor H-89 significantly inhibited M2 polarization but did not completely block it—suggesting that other pathways may act synergistically. Future studies should validate this by combining PKA and Epac pathway inhibition and detecting histone modification levels.

Animal experiments are critical for verifying the clinical relevance of molecular mechanisms. The nude mouse subcutaneous xenograft model showed that GJB2-KD significantly inhibited LUAD tumor growth, reducing tumor volume and weight, whereas GJB2-OE promoted progression. These results are consistent with in vitro experiments, providing in vivo evidence for the pro-tumor role of GJB2. At the molecular level, in vivo experiments further validated the integrity of the “GJB2-cAMP-PKA-CREB-M2 polarization” pathway: in the GJB2-KD group, nude mice showed reduced serum cAMP levels, decreased secretion of IL-10, TGF-β, and IL-4, increased M1 macrophage proportion and decreased M2 proportion in tumor tissues, and downregulated CREB phosphorylation, and PKA nuclear translocation. These findings are highly consistent with in vitro results, confirming the in vivo reliability of the mechanism.

Regarding the regulation of cAMP levels in LUAD cells, it should be clarified that the GJB2-KD/OE A549 and NCI-H1975 cells used in this study were cultured independently without THP-1 cell co-culture. Thus, the observed alterations in cAMP secretion are indicative of cell-autonomous regulatory mechanisms. Specifically, although GJB2 is classically recognized as a gap junction protein, accumulating evidence has demonstrated that connexins can also form functional hemichannels to mediate nucleotide release into the extracellular space. Connexin hemichannel-mediated ATP release can be hydrolyzed into adenosine, which in turn activates adenosine receptors coupled to adenylate cyclases (ACs), thereby enhancing intracellular cAMP production [23]. In addition, connexin proteins may indirectly modulate phosphodiesterase (PDE) activity through protein-protein interaction networks, although this hypothesis awaits direct validation in lung cancer models [24]. In the present study, the elevated cAMP secretion in GJB2-overexpressing LUAD cells is likely attributed to enhanced hemichannel-mediated ATP release and subsequent paracrine activation of ACs, whereas GJB2 knockdown attenuates this process. These cell-autonomous regulatory mechanisms enable LUAD cells to modulate the TME prior to the establishment of direct gap junctional communication with immune cells. Future studies should directly measure ATP release, adenosine levels, and AC/PDE activities in GJB2-manipulated LUAD cells to fully dissect the intricate regulatory pathways underlying this process.

In conclusion, this study is the first to confirm that GJB2 is highly expressed in LUAD and promotes LUAD progression by mediating cAMP transfer between tumor cells and macrophages, activating the PKA-CREB signaling pathway to induce M2 macrophage polarization. This discovery expands our understanding of the molecular regulatory mechanisms of LUAD and provides a novel biomarker for LUAD prognosis assessment and a potential target for targeted therapy—holding significant theoretical significance and clinical translational value.

Abbreviations

LUAD

Lung adenocarcinoma

KD

Knockdown

OE

Overexpression

EdU

5-ethynyl-2'-deoxyuridine

cAMP

Cyclic adenosine monophosphate

PKA

Protein kinase A

CREB

cAMP response element-binding protein

Authors’ contributions

Yuanhua Liu: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft. Guanghui Liu: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft. Jingjing Mei: Investigation, Validation, Data curation. Jia Li: Investigation, Validation, Resources. Jiming Si: Methodology, Investigation, Visualization. Yan Kang: Investigation, Visualization. Jianjun Jin: Conceptualization, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition.

Funding

This work was supported by Key scientific Research Project Plan of Colleges and universities of Henan Province (Grant numbers: 23A320014).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments conformed to the internationally accepted principles for the care and use of laboratory animals (Research and Clinical Trial Ethics Committee of the First Affiliated Hospital of Zhengzhou University).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.