CD4+ T Cell-Specific Proteomic Pathways Identified in Progression of Hypertension Across Postmenopausal Transition

Abstract

Background Menopause is associated with an increase in the prevalence and severity of hypertension in women. Although premenopausal females are protected against T cell-dependent immune activation and development of angiotensin II (Ang II) hypertension, this protection is lost in postmenopausal females. Therefore, the current study hypothesized that specific CD4⁺ T cell pathways are regulated by sex hormones and Ang II to mediate progression from premenopausal protection to postmenopausal hypertension. Methods and Results Menopause was induced in C57BL/6 mice via repeated 4-vinylcyclohexene diepoxide injections, while premenopausal females received sesame oil vehicle. A subset of premenopausal mice and all menopausal mice were infused with Ang II for 14 days (Control, Ang II, Meno/Ang II). Proteomic and phosphoproteomic profiles of CD4⁺ T cells isolated from spleens were examined. Ang II markedly increased CD4⁺ T cell protein abundance and phosphorylation associated with DNA and histone methylation in both premenopausal and postmenopausal females. Compared with premenopausal T cells, Ang II infusion in menopausal mice increased T cell phosphorylation of MP2K2, an upstream regulator of ERK, and was associated with upregulated phosphorylation at ERK targeted sites. Additionally, Ang II infusion in menopausal mice decreased T cell phosphorylation of TLN1, a key regulator of IL-2Rα and FOXP3 expression. Conclusions These findings identify novel, distinct T cell pathways that influence T cell-mediated inflammation during postmenopausal hypertension.

Keywords: 4‐vinylcyclohexene diepoxide; Ang II; T cells; menopause; phosphoproteomics; proteomics.

Figure 1. Ang II and Menopause exert distinct effects on CD4⁺ T cell protein profiles.

A, Splenic CD4⁺ T cells were isolated from Control, Ang II (800 ng/kg per minute, 14 days), and Meno/Ang II (VCD, 160 mg/kg, 20 days) female animals (n=4). CD4⁺ protein homogenates were separated into 8 lanes by SDS‐PAGE. Protein in each lane was excised and digested and the resulting peptides were analyzed by tandem mass spectrometry. Raw data processing for quantification was completed in Progenesis and peptide/protein identification was performed via alignment with Mascot database. Resulting Mascot peptide/protein identifications were imported into Progenesis for protein abundance quantification via extracted ion abundance. B, A total of 7085 proteins were identified and quantified, of which 546 were differentially expressed between the 3 groups (ANOVA, P≤0.05). C, Hierarchical protein clustering, heatmap visualization and protein cluster profiles of the 546 proteins were also performed in Perseus. This revealed 3 patterns of protein abundance in CD4⁺ T cell protein expression. D, Venn diagram of the pairwise (Student t test, P≤0.05) protein abundance differences between groups. A majority of protein abundance differences were observed between Control and Meno/Ang II CD4⁺ T cells. Ang II indicates angiotensin II; Meno, menopause; and VCD, 4‐vinylcyclohexene diepoxide.

Figure 2. In premenopausal T cells, Ang II increased abundance of proteins associated with transcription.

A, Heatmap visualization of the 384 (Student t test P≤0.05) significantly different proteins between Control and Ang II CD4⁺ T cells. The majority of proteins, 263, were upregulated with Ang II treatment. When Meno/Ang II protein abundance of the 384 proteins was observed, Meno/Ang II samples clustered more tightly with Ang II than Control samples. B, PANTHER classification of significantly overrepresented biological processes associated with upregulated (squares) and downregulated (circles) proteins in Ang II CD4⁺ T cells. C, PANTHER classification of significantly overrepresented molecular functions associated with upregulated (squares) proteins in Ang II CD4⁺ T cells. No molecular functions were significantly overrepresented amongst downregulated proteins. D, Volcano plot identification of upregulated (purple) and downregulated (green) proteins in Ang II CD4⁺ T cells, P≤0.05, log2 fold change > |1|. The 4 proteins with the greatest upregulation and downregulation, as determined by Euclidean distance from the origin (0,0), were BRNP3, DAXX, CDK12, SENP8 and MPEG1, OXLA, CATR, SERC1 respectively. E, Pscan identification of transcription factors significantly associated with promoter regions (−450 to +50) of upregulated (purple) and downregulated (green) proteins in Ang II CD4⁺ T cells. Ang II indicates angiotensin II; BRNP3, BMP/retinoic acid‐inducible neural‐specific protein 3; CATR, cathepsin R; CDK12, cyclin‐dependent kinase 12; DAXX, death domain‐associated protein 6; Meno, menopause; MPEG1, macrophage‐expressed gene 1 protein; OXLA, L‐amino‐acid oxidase; SENP8, sentrin‐specific protease 8; and SERC1, serine incorporator 1.

Figure 3. In menopausal T cells, Ang II differentially regulated KLF14 transcription.

A, Heatmap visualization of the 285 (Student t test P≤0.05) significantly different proteins between Ang II and Meno/Ang II CD4⁺ T cells. The majority of proteins, 189, were downregulated in Meno/Ang II. When Control protein abundance of the 285 proteins was observed, Ang II and Control samples clustered. B, PANTHER classification of overrepresented biological processes associated with upregulated (squares) and downregulated (circles) proteins in Meno/Ang II CD4⁺ T cells. C, PANTHER classification of significantly overrepresented molecular functions associated with upregulated (squares) and downregulated (circles) proteins in Meno/Ang II CD4⁺ T cells. D, Volcano plot identification of upregulated and downregulated proteins in Meno/Ang II CD4⁺ T cells: the 4 proteins with the greatest upregulation and downregulation were COX17, OXLA, RRP36, EZH1 and MYCN, OASL2, GPAA1, COR2A, respectively. E, Pscan promoter region analysis of upregulated and downregulated proteins predicted KLF14 binding in both subsets of proteins. Ang II indicates angiotensin II; COR2A, Coronin‐2A; COX17, Cyctochrome c oxidase copper chaperone; EZH1, Histone‐lysine N‐methyltransferase 1; GPAA1, glycosylphosphatidylinositol anchor attachment 1 protein; KLF14, Krueppel‐like factor 14; Meno, menopause; MYCN, N‐myc proto‐oncogene protein; OASL2, 2’‐5’‐oligoadenylate synthase‐like protein 2; OXLA, L‐amino‐acid oxidase; and RRP36, ribosomal RNA processing protein 36 homolog.

Figure 4. Phosphoproteome analysis of CD4⁺ T cells uncovered distinct effects of both Ang II and menopause.

A, Progenesis was used to identify peptide ion modifications and quantify extracted phosphopeptide ion abundance. In total, 6059 phosphosites within CD4⁺ T cell proteins were identified, of which 466 phosphosites on 224 proteins were differentially phosphorylated (ANOVA, P≤0.05) between the 3 groups. B, Hierarchical clustering confirmed distinct treatment effect on CD4⁺ T cell protein phosphorylation. Heatmap and cluster profile visualization identified 3 patterns of phosphorylation. C, Venn diagram of the pairwise (Student t test, P≤0.05) peptide phosphorylation differences between groups. A majority of differences were observed between Control and Meno/Ang II CD4⁺ T cells. Ang II indicates angiotensin II; and Meno, menopause.

Figure 5. Ang II increased proline‐directed protein phosphorylation linked to chromatin regulation by methylation.

A, Heatmap visualization of the 226 (Student t test P≤0.05) significantly different phosphosites between Control and Ang II CD4⁺ T cells. Phosphorylation of the majority of phosphosites, 200, was upregulated with Ang II treatment. When Meno/Ang II phosphorylation was observed, Meno/Ang II samples clustered more tightly with Ang II than Control samples. B, PANTHER classification of significantly overrepresented biological processes associated with proteins with upregulated phosphorylation in Ang II CD4⁺ T cells. C, PANTHER classification of significantly overrepresented molecular functions associated with proteins with upregulated phosphorylation in Ang II CD4⁺ T cells. No significantly overrepresented biological processes or molecular functions were associated with proteins with downregulated phosphorylation. D, Volcano plot identification of upregulated (purple) and downregulated (green) phosphosites of interest in Ang II CD4⁺ T cells. The 4 phosphosites with the greatest upregulation and downregulation were FBRL S130, IWS1 S666, PININ S346, DAXX S515 and BNIP2 S114, HNRPF Y276, HS90A S252, HCLS1 S333, respectively. E, IceLogo determination of overrepresented amino acid residues surrounding (±6 AAs) significantly downregulated phosphosites. Kinases predicted (Scansite ver. 4.0) to phosphorylate downregulated phosphosites. F, IceLogo determination of overrepresented amino acid residues surrounding significantly upregulated phosphosites along with kinases predicted to regulate upregulated phosphosites. Ang II indicates angiotensin II; BNIP2, BCL2/adenovirus E1B 19 kDa protein‐interacting protein 2; DAXX, death domain‐associated protein 6; FBRL, rRNA 2’‐O‐methyltransferase fibrillarin; HCLS1, hematopoietic lineage cell‐specific protein; HNRPF, heterogeneous nuclear ribonucleoprotein F; HS90A, heat shock protein HSP 90‐alpha; IWS1, protein IWS1 homolog; Meno, menopause; and PININ, Pinin.

Figure 6. Meno/Ang II resulted in decreased proline‐directed protein phosphorylation, notably S405 within TLN1, a positive regulator of Treg maintenance.

A, Heatmap visualization of the 207 (Student t test P≤0.05) significantly different phosphosites between Ang II and Meno/Ang II CD4⁺ T cells. Phosphorylation of the majority of phosphosites, 115, was downregulated after menopause induction. When Control phosphorylation was observed, Control and Ang II samples clustered. B, PANTHER classification of significantly overrepresented biological processes associated with proteins with downregulated phosphorylation in Meno/Ang II CD4⁺ T cells. No significantly overrepresented biological processes were associated with proteins with upregulated phosphorylation. C, PANTHER classification of significantly overrepresented molecular functions associated with proteins with downregulated and upregulated phosphorylation in Meno/Ang II CD4⁺ T cells. D, Volcano plot identification of upregulated (purple) and downregulated (green) phosphosites in Meno/Ang II CD4⁺ T cells. The 4 phosphosites with the greatest upregulation and downregulation were PRP4B S278, MP2K2 S23, PHAR4 S118, CP100 S244 and EIF3B S120, MYCN S350, TOE1 S349, and TLN1 S405, respectively. E, IceLogo determination of overrepresented amino acid residues surrounding (±6 AAs) significantly downregulated phosphosites. Kinases predicted (Scansite ver. 4.0) to phosphorylate downregulated phosphosites. F, IceLogo determination of overrepresented amino acid residues surrounding significantly upregulated phosphosites along with kinases predicted to regulate upregulated phosphosites. Ang II indicates angiotensin II; CP100, cilia‐ and flagella‐associated protein 100; EIF3B, eukaryotic translation initiation factor 3 subunit B; Meno, menopause; MP2K2, dual specificity mitogen‐activated protein kinase kinase 2; MYCN, N‐myc proto‐oncogene protein; PHAR4, phosphatase and actin regulator 4; PRP4B, serine/threonine‐protein kinase PRP4 homolog; TLN1, talin‐1; and TOE1, target of EGR1 protein 1.

Figure 7. Proposed T cell mechanisms involved in postmenopausal hypertension.

Compared with CD4⁺ T cells from Ang II infused premenopausal females, CD4⁺ T cells from Ang II infused menopausal mice demonstrated increased expression or phosphorylation (solid purple) and predicted activity (striped purple) of proteins associated with decreased FOXP3 and IL‐10 transcription and increased Th17 cell development. Further, CD4⁺ T cells from Ang II infused menopausal mice demonstrated decreased (green) S405 phosphorylation of TLN1, a positive regulator of Treg function. Ang II indicates angiotensin II; ERK, mitogen‐activated protein kinase; EZH1, histone‐lysine N‐methyltransferase 1; FOXP3, foxhead box protein P3; IFNγ, interferon gamma; IL‐10, interleukin‐10; IL‐2R, interleukin‐2 receptor; KLF14, Krueppel‐like factor 14; MP2K2, dual specificity mitogen‐activated protein kinase kinase 2; OXLA, L‐amino‐acid oxidase; RORγT, retinoic acid receptor‐related orphan nuclear receptor gamma‐thymic; STAT5, signal transducer and activator of transcription 5; and TLN1, talin‐1.