Ethanol inhibited growth hormone receptor-mediated endocytosis in primary mouse hepatocytes

Introduction

Approximately 2 billion people worldwide drink alcoholic beverages. Moderate wine consumption is reported to reduce the risk of developing Alzheimer’s disease and has hepatoprotective function [1], [2], [3]. However, long-term or heavy drinking clearly leads to liver damage [4]. The mechanism underlying this phenomenon is still not comprehensively understood. Several studies have reported that alcohol have harmful effects on body via inducing significant changes in cell metabolism and signal transduction [5]. As early as in the 1980s, it was discovered that alcohol can impair receptor-mediated endocytosis (RME) [6]. Subsequently, multiple pathways by which alcohol affects RME were discovered. For example, alcohol can affect the internalization of epidermal growth factor (EGF) by diminishing the number of cell surface receptors [7]. It can affect the internalization of envelope protein gp120 of human immunodeficiency virus 1 in hepatocytes though the lysosome/ubiquitin proteolytic pathways [8]. Moreover, it can change the fluidity of plasma membrane through the cholesterol in the plasma membrane and manage the intracellular distribution of Rab protein to affect the clathrin-independent endocytosis [9]. Alcohol increases the level of Arf6 in the nervous system, which controls the assembly of AP-2/clathrin coat in clathrin-mediated endocytosis [10].

Growth hormone (GH) is a 191‐amino acid polypeptide hormone. GH can serve various physiological functions including regulate the metabolism of proteins, lipids, and carbohydrates [11]. GH exerts its biological effects via binding to the growth hormone receptor (GHR) [12]. GHR is a class-I cytokine receptor containing 638 amino acids. It is widely expressed as a dimer on the body’s cell membrane and greater levels in the liver. GH can activate the extracellular domain (EDC) of GHR, which in turn activates Janus kinase2 (JAK2). After this, a number of down-stream intracellular signaling molecules, including signal transducers and activators of transcription (STAT; STATs 1/3/5), are phosphorylated to enable GH to perform related biological functions [13], [14], [15], [16].

Internalized GH and GHR exist in the form of GH-GHR dimers. It has been reported that ethanol suppresses the GHR signaling rather than ligand binding [17], 18]. The nuclear localization of GHR-dependent GH is crucial for initiating various GH-induced signal transduction pathways [19], [20], [21]. Endocytosis is a process necessary for many important physiological functions of cells. The intracellular endocytosis occurs via multiple pathways. For instance, caveolin‐dependent, clathrin‐dependent, and caveolin‐ and clathrin‐independent. Ethanol could inhibit caveolin-dependent or clathrin- and caveolin-independent endocytosis by impairing asialoglycoprotein receptor function, disturbing protein trafficking [22], 23]. Moreover, ethanol impairs receptor-mediated endocytosis of various proteins, including EGFR, via some endocytosis defect or receptor recycling [7], [24], [25], [26]. However, whether ethanol affects GHR endocytosis has yet to be elucidated.

In the present study, we investigated the molecular mechanism that ethanol inhibits GH biological activity from the perspective of cell internalization. We initially explored the impact of ethanol on GH-induced intracellular signalling by Western blot analysis. Furthermore, we determined the effect of ethanol on GH-GHR’s nuclear localization and endocytosis by indirect immunofluorescence and colocalization experiments. We postulated that ethanol may inhibit the endocytosis mediated by GHR. This study reported a new mechanism via which ethanol inhibits the biological activity of GH.

Materials and methods

Results

GHR expression on mouse hepatocytes

First, we examined the GHR expression on hepatocytes. As depicted in Figure 1, GHR was primarily found in the cell membrane and cytoplasm. The nucleus was stained blue by DAPI. The control antibody did not demonstrate any positive staining (data not presented).

Figure 1:

CLSM analysis to evaluate the expression of GHR on hepatocytes. After rinsing, the cells were, fixed, then blocked with BSA. After washing with PBS, the cells were incubated with anti-GHR antibodies at 4 °C overnight. Then the cells were incubated with secondary. The cell nuclei were stained with DAPI. The cell samples were visualized by CLSM. Bar=10 μm. The figures shown represent at least three separate experiments.

GHR-mediated intracellular signaling is suppressed by ethanol

The endogenous expression of GHR in the hepatocytes were assessed via western blotting. The results showed that GHR expression decreased after GH treatment in a time‐dependent manner (Figure 2A). Furthermore, the impact of ethanol on GH-induced intracellular signaling was assessed. When the cells were treated with GH or EtOH+GH, the phosphorylation levels of intracellular signaling proteins (JAK2, and STAT1/3/5) first increased and then decreased. Ethanol strongly reduced the phosphorylation levels of these signaling proteins (Figure 2B–E). These findings indicated that ethanol inhibited GHR-mediated intracellular signaling in a time-dependent manner.

Figure 2:

The impact of EtOH on GHR-mediated intracellular signaling proteins. (A) Serum-starved mouse hepatocytes were treated with GH (20 μg/mL) for various time periods (0, 15, 30, 60, and 90 min). Then, the expression levels of GHR protein were established via western blot. (B–E) effect of ethanol on GH induced intracellular signal transduction. The expression of phosphorylated and total JAK2 (B), STAT1 (C), STAT3 (D), and STAT5 (E) was detected by western blot. The figures shown represent at least three separate experiments. *p<0.05, **p<0.01 compared with the GH group.

Internalization and nuclear localization of GH-GHR with or without ethanol stimulation

To observe the internalization of GH after ethanol stimulation, cells were treated with GH or EtOH+GH (Figure 3A). The IFA experiments indicated that amounts of GH (green) were observed in the nucleus in the control group. And the nuclear localization of GH was significantly reduced after ethanol stimulation compared with GH alone. Following ethanol treatment, we also examined GHR expression in the cell membrane and nucleus using Western blot analysis. Figure 3B illustrates that upon GH administration, GHR was present in both the nuclear extract and the cytoplasm. However, after exposure to ethanol, GHR was primarily detected in the cytoplasm, with minimal expression observed in the nucleus. These results illustrated that ethanol may inhibit the nuclear translocation of GH.

Figure 3:

The impact of EtOH on the nuclear localization of GH in mouse hepatocytes. (A) The cells were exposed to alexa fluor 488-GH (20 μg/mL) for 30min, or exposed to alexa fluor 488-GH (20 μg/mL) after EtOH (1 % v/v) treated for 30min. After washing, the cells were fixed and stained with DAPI (nucleus). The cells were subjected to CLSM. Bar=10 μm **p<0.01 compared with the GH group. (B) The expression of GHR in the membrane and nuclei of mouse hepatocytes. The cells were exposed to GH (20 μg/mL) for 30 min, or exposed to GH (20 μg/mL) after EtOH (1 % v/v) treated for 30min, the expression levels of GHR protein in the membrane and nuclei were detected by western blot. The figures shown represent at least three separate experiments.

Ethanol inhibited the nuclear localization of GH in a time‐dependent manner

CLSM was employed to investigate the impact of ethanol on the internalization of GH in the hepatocytes at various times (5–90 min). Without ethanol treatment, a substantial quantity of GH (green) was located on the plasma membrane and in the cytoplasm and nucleus in 30–60 min (Figure 4). However, subsequent to ethanol treatment, GH was found to be mostly present on the plasma membrane with minimal presence within the cell nucleus. The findings indicated that ethanol suppresses the nuclear localization of GH in a time-dependent manner.

Figure 4:

The dynamics of GH internalization after stimulation with GH or EtOH+GH. The cells were exposed to alexa fluor 488-GH (20 μg/mL), or exposed to alexa fluor 488-GH (20 μg/mL) after EtOH (1 % v/v) treated for different durations. The cells were fixed with 4 % paraformaldehyde. After washing with PBS, the cells were visualized using CLSM. Bar=10 μm. The figures shown represent at least three separate experiments. Distinct letters indicate a statistically significant difference (p<0.05).

GH‐GHR endocytosis is affected by ethanol

The endocytosis pathway mediated by clathrin and cavoelin is reported to have a vital function in the internalization of GHR. To identify whether ethanol affects GH-GHR internalization, colocalization experiments were conducted using CLSM. The presence of cavoelin and GHR colocalization signals was identified when the cells were exposed alone to GH (Figure 5). However, after treating both ethanol and GH, the colocalization signals of GHR and cavoelin markedly decreased in the nucleus. The results suggesting that ethanol can not only inhibit GH-mediated intracellular signaling at the plasma membrane but can also affect GH‐GHR endocytosis (summarized mechanisms are depicted in Figure 6).

Figure 5:

The endocytosis of GH or EtOH+GH. The cells were exposed to GH (20 μg/mL) for 30 min, or exposed to GH (20 μg/mL) after EtOH (1 % v/v) treated for 30min, after which the cells were washed three times. Then cells were further fixed with 4 % paraformaldehyde, then blocked with 3 % BSA, and immunostained utilizing anti‐cavoelin (green), and anti‐GHR antibodies (red). Following the last round of washing, the cell samples were visualized using CLSM. Bar=10 μm. The figures shown represent at least three separate experiments.

Figure 6:

Possible mechanisms by which ethanol inhibits the biological activity of GH. GH binding with GHR can not only activate GHR-mediated intracellular signaling but also lead to GHR transport into nucleus. In traditional research, ethanol inhibits the biological activity of GH by inhibiting GHR-mediated signal transduction. The present study found a novel possible molecular mechanism: ethanol could inhibit the biological activity of GH via inhibiting GHR-mediated endocytosis, which may be related to the inhibition of the interaction between GHR and caveolin. This study laid a foundation for further exploring the mechanism underlying the effects of ethanol on GH biological activity.

Discussion

It is well known that GH can modulate various metabolic processes, and the suppression of GH-mediated intracellular signaling by ethanol may be a contributing factor to the onset of numerous ethanol-related diseases. Previous studies have reported that ethanol can inhibit spontaneous pulsatile GH secretion [17]. Ethanol can inhibit GH-induced protein synthesis without altering the number or binding characteristics of GHR. Traditional views suggest that ethanol inhibits the biological activity of GH by inhibiting GH-induced signal transduction [18]. However, the precise mechanism responsible for this inhibition is still not completely comprehended. In this investigation, to our knowledge, we present the first exploration of the possible molecular mechanism that ethanol could inhibit biological activity from the perspective of cell internalization.

Our experimental results indicate that GHR was abundantly expressed in the cytoplasm and on the cell membrane. According to the previous studies, JAK2 and GHR are tyrosine-phosphorylated subsequently, and down-stream signaling is subsequently activated after GH interacts with GHR on the cell membrane [13], [14], [15], [16]. In the current study, we found that ethanol inhibited GHR-mediated phosphorylation levels of JAK2 and STAT1/3/5 proteins in a time-dependent manner. However, it has been reported that GH not only displays physiological roles at the cell membrane but also exhibits physiological functions in the cytoplasm and cell nuclei [20], 29].

Internalization is the starting step of nuclear transport of the GH-GHR complex. After binding with GHR, intracellular signaling molecules are activated, and the internalization process of the GH-GHR complex is also initiated. Nuclear localization of GH-GHR can still exhibit important biological activities [20]. Thus, we investigated how ethanol influences the activity of GH-induced intracellular signaling molecules by focusing on the process of cell internalization. It was observed that ethanol suppressed the nuclear localization of GH-GHR. Furthermore, it was noted that following GH stimulation, GH-GHR underwent rapid nuclear translocation in a time-dependent manner, and ethanol blocked the nuclear localization of GH-GHR. This suggested that ethanol may inhibit GH signaling ability through inhibiting GHR-mediated nuclear translocation.

Lipid rafts/vesicles are used as signal transduction platforms by many membrane proteins, mediating important physiological processes [30]. Alcohol can damage the lipid rafts/vesicles of membrane proteins. High concentrations of alcohol can induce complete translocation of these membrane proteins from lipid rafts/vesicles, leading to various pathological changes in various tissues [31]. Strous and Lobie et al. reported that the GH-GHR complex mainly enters the cytoplasm through internalization mediated by clathrin and caveolin [32]. Dalke et al. reported that after long-term alcohol intake, EGFR-mediated endocytosis in rat liver cells was inhibited, affecting multiple parts of the body including surface receptor expression, internalization, intracellular transport to lysosomes, and receptor recycling [7]. Long-term consumption of alcohol can damage protein transport and selectively damage receptor-mediated endocytosis [33]. In our study, GH-GHR could enter into the cytoplasm via caveolin-mediated endocytosis. However, the colocalization signals of GHR-cavoelin were significantly reduced in the nucleus when the cells were exposed to with ethanol and GH. These findings indicate that ethanol may inhibit GHR-mediated nuclear translocation through inhibiting caveolin-dependent endocytosis. However, in addition to caveolin-mediated endocytosis, endocytosis is inhibited in cells via many ways. Thus, we are unable to exclude additional unidentified factors.

In conclusion, we explored the mechanisms underlying the inhibition of GHR-mediated signaling by ethanol in primary mouse hepatocytes. Ethanol could inhibit the nuclear localization of GH-GHR, which may be linked to the inhibition of the interaction between GHR and caveolin. The combined effect of these factors downregulated the GH-GHR signal. Thus, we postulated that ethanol inhibits the endocytosis mediated by GHR. The groundwork for further investigation into the mechanism behind ethanol’s impact on GH biological activity was established by this study. Nevertheless, further research should be done to identify the precise mechanism by which ethanol impacts the biological action of GH.