Cortisol-Responsive regulation of RASGRP1 in lymphoblastoid cells from individuals with bipolar disorder

Abstract

Objective

Bipolar Disorder (BD) is a complex psychiatric disorder, characterised by recurrent episodes of mania and depression, and is associated with the dysregulation of stress hormone signalling, immune and inflammatory pathways. However, limited information is currently available on the effects of stress hormones on immune and inflammatory pathways in BD. Ras guanyl releasing protein 1 (RASGRP1) is a pro-inflammatory guanine nucleotide exchange factor involved in Ras-MAPK signalling. Cortisol exerts well-known anti-inflammatory effects; we therefore hypothesized that cortisol exposure would regulate RASGRP1 expression. This study, therefore, examined the impact of cortisol on RASGRP1 transcriptional and translational expression across BD subtypes in lymphoblastoid cell lines derived from BD and non-BD individuals.

Results

We observed differential responses to cortisol among the cell lines. Specifically, at the level of transcription expression was elevated in BD II cortisol-treated cells at basal levels vs. stress induced levels of cortisol. Concurrently, RASGRP1 protein was detected only in BD II cells treated with basal levels of cortisol. No protein was detected in untreated cells or those exposed to stress-induced levels of cortisol.

Conclusion

RASGRP1 expression is responsive to cortisol in a concentration-dependent manner.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13104-026-07898-0.

Introduction

Bipolar Disorder (BD) is a complex mood disorder characterised by recurrent episodes of mania or hypomania and depression, with BD I involving at least one manic episode and BD II defined by hypomania and major depression [1]. BD affects approximately 1–2% of the global population and is associated with substantial functional impairment and elevated suicide risk [2]. Although mood stabilisers, antipsychotics, and adjunctive antidepressants are widely used, many patients experience incomplete symptom control, adverse effects, and frequent relapse [3]. These limitations reflect an incomplete understanding of the biological mechanisms underlying BD, including the pathways linking mood instability, stress sensitivity and inflammatory and immune dysregulation.

Immune dysregulation and neuroinflammatory mechanisms are increasingly recognised in BD pathophysiology; however, the contribution of intracellular signalling regulators to these processes is poorly understood. Elevation of multiple pro-inflammatory cytokines has been observed during manic and depressive episodes [4]. These cytokines influence neuroinflammation and neurotransmission by reducing serotonin, damaging mitochondrial function, impairing neurogenesis, and inducing oxidative and nitrosative stress [5]. These downstream effects reflect the impact of sustained inflammatory signalling. Immune alterations may result from interactions between environmental factors such as sleep and circadian disruptions, chronic stress, autoimmune responses, and genetic vulnerability [6]. For example, variants in genes involved in neuronal ion channel regulation, such as calcium voltage-gated channel subunit alpha1 C (CACNA1C) which is crucial for calcium transport in the brain and Ankyrin 3 (ANK3) which regulates neuronal excitability are associated with BD. This further supports the role of dysregulated signalling pathways in BD, highlighting the need to investigate intracellular regulators that integrate inflammatory and neuronal pathways, rather than focusing solely on cytokines [6].

RASGRP1 is a diacylglycerol and calcium-regulated guanine nucleotide exchange factor that activates the Ras-MAPK signalling pathway. It plays a key role in lymphocyte development and immune activation [7]. Beyond immunity, RASGRP1 contributes to neuronal signalling by modulating Ras-dependent synaptic plasticity and intracellular calcium responses [8, 9]. Altered RASGRP1 expression has been implicated in disorders such as Parkinson’s disease and schizophrenia [7, 10]. Additionally, peripheral studies using lymphoblastoid cell lines (LCLs) report reduced RASGRP1 expression in BD [11].

Furthermore, RASGRP1 interacts with Rhes, a protein necessary for motor and neuronal function. Additionally it is regulated by the transcription factor Nurr1, a dopaminergic and nuclear receptor with anti-inflammatory activity [12, 13]. These observations suggest that RASGRP1 may occupy a biologically relevant intersection between immune and neuronal pathways in BD.

Cortisol, the primary glucocorticoid released in response to stress, influences immune activity, inflammatory signalling, and cellular stress responses in BD [14, 15]. Specifically, studies show Ras-MAPK signalling, a pathway implicated in BD is altered by cortisol [8, 9, 16]. Since RASGRP1 is a critical activator of Ras-MAPK signalling, characterizing its regulation is an important step to understanding the underlying mechanisms of BD. In the current study, we hypothesized that cortisol would regulate RASGRP1 expression. This may provide insight into a possible link between immune dysregulation, inflammation, and neuronal stress pathways in BD [17].

To test our hypothesis, we characterised the effect of cortisol on RASGRP1 expression in lymphoblastoid cell lines (LCLs) derived from individuals diagnosed with BD I and BD II. LCLs provide a tractable and cost-effective model to examine how stress hormones influence gene regulation outside the brain [11].

Methods

LCLs derived from individuals with BD I, BD II, and non-BD (NBD) controls were obtained from the Coriell Institute and cultured in RPMI 1640 supplemented with 20% FBS and 5% penicillin/streptomycin at 37 °C with 5% CO₂. Cells were seeded at 400,000 cells/ml and treated for 48 h with 10ng/ml or 100ng/ml cortisol to represent basal and stress conditions [18].

RNA extraction and RT-qPCR

Total RNA was extracted using the Aurum RNA Mini Kit with on-column DNase treatment, and RNA concentration and integrity were verified by spectrophotometry and agarose gel electrophoresis. cDNA was synthesised using the iScript kit, and optimal RNA input was confirmed through agarose gel analysis and determined to be 1𝔲g. RASGRP1 mRNA expression was quantified using SYBR Green based qPCR with melt curve analysis and gel electrophoresis to confirm specificity. Relative expression was calculated using the Livak ΔΔCq method [19] with RPS17 as the reference gene.

Protein extraction and western blot

For protein analysis, cells were lysed in RIPA buffer, and protein concentrations were measured using the BCA assay. Equal amounts of protein (20 µg) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with RASGRP1 primary antibody (1:1000) and HRP-conjugated secondary antibody (1:20,000), with detection on a LICOR C-Digit scanner.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10. A two-way ANOVA and Tukey’s post hoc test were used for cortisol and subtype comparisons, as well as their interaction. Comparisons between BD and NBD groups were performed using the Mann-Whitney U test. All data are presented as mean ± SEM and significance was set as p < 0.05.

Results

Relative RASGRP1 expression

RASGRP1 was increased in BD I by 1.3-fold and decreased in BD II (0.9-fold) relative to NBD controls. No significant difference was observed in the expression of RASGRP1 in BD I, BD II, and NBD cell lines after analysis with a Mann-Whitney U test (p = 0.3429, U = 4) (Fig. 1).

Fig. 1.

Relative RASGRP1 expression in BD I and BD II LCLs compared to NBD LCLs. Values representing mean ± SEM (standard error of mean) The 2-ΔΔCq method was used to determine fold change. Broken lines represent NBD expression. p = 0.3429 by Mann-Whitney U test (n = 4)

Effect of cortisol on RASGRP1 transcription in BD LCLs

A two-way ANOVA (treatment by subtype) revealed a significant interaction effect on RASGRP1 expression (Table 1). Specifically, RASGRP1 expression at basal level cortisol (10ng/ml), in the BD II subtype, exhibited a 0.7-fold higher expression compared to BD I (p = 0.006). And BD II exhibited a 0.9-fold higher expression compared to NBD (p = 0.002). Additionally, within the NBD subtype, no treatment resulted in a 1.5-fold greater expression compared to basal (p = 0.013) and 1.4-fold greater expression compared to stress-induced levels (p = 0.017).

Table 1.

Two-way ANOVA summary for the effect of cortisol on RASGRP1 expression in BD I, BD II and NBD LCLs ^a

Source of variation	SSb	DFb	Fb	P valueb
Subtype x Treatment	8.130	4	4.5	0.0062
Subtypes	1.850	2	2.0	0.1460
Treatment	3.993	2	4.4	0.0212
Residual	12.08	27	-	-

^aData are original and derived from two-way ANOVA of the effect of Cortisol on RASGRP1 expression

^bSS sum of squares; DF Degrees of freedom; F F-statistic; P value probability value

Relative gene expression was calculated using the 2^− ΔΔCq and normalised to the no treatment condition within each cell line (Fig. 2).

Fig. 2.

Effect of Cortisol on RASGRP1 expression in BD I, BD II and NBD LCLs. Statistical analysis between groups & treatments were analysed using a two-way ANOVA followed by Tukey’s post hoc tests. ** p < 0.01, * p < 0.05. n = 5

Effect of cortisol on RASGRP1 translation in BD LCLs

The RASGRP1 protein was detected at approximately 110–120 kDa only in BD II LCLs grown in basal levels cortisol. No protein was detected at stress-induced levels of cortisol in BD I or NBD LCLs. (Fig. 3). The higher than expected molecular weight (90–100 kDa) is consistent with post-translational modifications (phosphorylation) observed in RASGRP1 [20].

Fig. 3.

RASGRP1 protein expression in BD. Representative blot (n = 2) showing expression of RASGRP1 in BD - derived LCLs grown in the absence (no treatment) and presence of basal (10ng/ml) and stress-induced (100ng/ml) levels of cortisol. Western blot analysis with RASGRP1 polyclonal antibody (1:1000) and HRP-conjugated secondary antibody (1:20,000)

Discussion

This study compared the effects of cortisol on RASGRP1 expression at the transcriptional and translational levels in BD I, BD II, and NBD LCLs. Our results showed that, at the level of transcription, RASGRP1 expression in the NBD and BD I cell lines decreased in the presence of both basal and stress-induced cortisol levels, whereas it increased significantly in the BD II cell lines at basal cortisol levels. RASGRP1 protein expression appeared to align with mRNA expression, as it was detectable only in BD II cell lines at basal cortisol levels. Additionally, no significant difference in RASGRP1 expression between BD subtypes, nor between the BD cell lines and the NBD control at either level of expression.

This study represents an early attempt to examine RASGRP1 expression across subtypes of bipolar disorder. A previous study [11] found reduced RASGRP1 expression in BD-derived LCLs compared to NBD control. However, that study analysed BD subtypes collectively, which may explain the discrepancy with our findings, in which no significant difference in RASGRP1 expression was observed between the BD and NBD groups.

Cortisol treatment revealed distinct, dose-dependent changes in RASGRP1 expression across the three cell lines. While RASGRP1 expression decreased in BD I and NBD groups with increasing cortisol exposure, BD II cells showed increased expression at the basal level of cortisol. This suggests that BD II cells may have differential sensitivity to mild glucocorticoid stimulation, despite cortisol’s anti-inflammatory functions. The increase at basal-level, contrasts with the decrease at stress-induced level of cortisol, which aligns with higher dose glucocorticoid suppression. These patterns support disrupted glucocorticoid signalling in BD [18] and may indicate altered glucocorticoid receptor feedback or desensitisation similar to findings reported by Belvederi Murri, Prestia [21]. Decreased expression across cortisol treatments in NBD cells further suggests that RASGRP1 is inherently responsive to glucocorticoids even in the absence of psychiatric pathology.

At the protein level, RASGRP1 was detectable only in BD II LCLs exposed to basal cortisol levels, partially mirroring the mRNA findings. The absence of protein in the remaining treatment groups may reflect post-transcriptional regulation, translational control, protein instability, or threshold-dependent expression. This selective protein detection suggests that low-dose cortisol may create a functional window that permits both transcription and translation of RASGRP1 in BD II cells. These findings are consistent with evidence that RASGRP1 is an inducible protein that participates in inflammatory Ras-ERK pathways, in which its expression and activity are sensitive to glucocorticoid- and cytokine-regulated signalling environments [22, 23]. RASGRP1’s regulation by neuroprotective factor Nurr1, which suppresses inflammatory gene expression [23], further supports the idea that RASGRP1 protein expression may depend on the balance between stress-hormone signalling and inflammatory signalling.

The differential response to glucocorticoid stimulation between BD I and BD II hints at subtype-specific molecular alterations, supporting growing evidence that BD symptoms subtypes may differ biologically and not only clinically [24]. These subtype-specific molecular differences could prove valuable for diagnosis, potentially serving as a peripheral biomarker for subtype distinction and facilitating earlier, more accurate identification of BD II, a subtype often misdiagnosed as unipolar depression [24, 25]. Beyond diagnostics, this differential stress sensitivity profile may carry meaningful clinical utility, as a deeper understanding of the molecular mechanisms underlying BD could inform the development of more personalised, subtype-tailored treatment strategies.

Limitations

Although these findings offer some insights, several limitations should be considered. The use of LCLs, while practical for peripheral gene expression studies, may not fully replicate in vivo neuronal behaviour. In addition, the limited sample size may restrict the extent to which these findings can be generalised to broader populations.

Conclusion

This study offers preliminary evidence that RASGRP1 expression is modulated by cortisol in a subtype- and dose-dependent, non-linear manner. These findings also support the use of RASGRP1 as a potential biomarker of stress sensitivity and immune dysregulation in BD, particularly BD II, thereby linking glucocorticoid signalling and inflammation in psychiatric disorders.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

BD

Bipolar disorder

RASGRP1

Ras guanyl releasing protein 1

LCLs

Lymphoblastoid cell lines

NBD

Non bipolar disorder

BD I

bipolar disorder I

BD II

Bipolar disorder II

Ras-MAPK

Ras mitogen activated protein kinase

Ras-ERK

Ras Extracellular signal-regulated kinase

CACNA1C

Calcium Voltage-Gated Channel Subunit Alpha1C

ANK3

Ankyrin 3

qPCR

quantitative polymerase chain reaction

BCA

Bicinchoninic acid

SDS-PAGE

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

PVDF

Polyvinylidene fluoride

Author contributions

Study conceptualisation and design: SS, MM, Investigation: SS, Statistical analysis: SS, Writing: SS, Review and editing: MM, Funding Acquisition: SS, MM.

Funding

This work was funded by Andrews University Office of Research.

Data availability

The data used and/or analysed in this study is available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.