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

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.

angiotensin II

cyclin‐dependent kinase

mitogen‐activated protein kinase

enhancer of zeste homolog

trans‐acting T‐cell‐specific transcription factor GATA‐3

glycogen synthase kinase 3

histone 3 trimethylation of the 27th lysine

histone H4 transcription factor

high salthigh salt

Krueppel‐like factor 14

dual specificity mitogen‐activated protein kinase kinase 2 (aka MEK2)

macrophage‐expressed gene 1 protein

recombination activating gene‐1

talin‐1

T regulatory cell

4‐vinylcyclohexene diepoxide

Clinical Perspective

What Is New?

To our knowledge, this is the first proteomic study of T‐cell specific pathways in pre and postmenopausal females to establish that Ang II infusion and a menopausal increase in blood pressure have distinct effects on the CD4+ T cell proteome and phosphoproteome.

The identified T cell molecular pathways are hypothesized to be involved in premenopausal protection against hypertension and menopausal susceptibility to hypertension development.

What Are the Clinical Implications?

The differentiation of CD4+ T cells has previously been implicated in sex differences in hypertension development; thus far the effect of sex hormones on CD4+ T cell pathways has not been studied.

The current study used a proteomic and phopshoproteomic approach to identify novel regulated CD4+ T cell pathways which are altered following Ang II infusion.

We found that in the context of Ang II infusion, menopause is associated with an increased ERK activity and reduced activity and expression of Treg associated pathways, suggesting an increased Th17/Treg ratio.

Treg associated pathways, such as TLN1 phosphorylation, are potential therapeutic targets for postmenopausal hypertension.

Hypertension is the leading risk factor for global disease burden. While premenopausal females are protected from hypertension, about 40% of a woman’s lifespan is spent in a postmenopausal state that is associated with increased hypertension and cardiovascular disease risk. , , Mechanistic understanding of this shift in blood pressure regulation is inadequate and correlates to greater uncontrolled hypertension in postmenopausal women compared with age matched men, despite greater medication adherence. , Using the VCD‐menopause model (VCD, 4‐vinylcyclohexene diepoxide), a novel estrogen‐deplete, ovary‐intact menopause model, , we have shown that premenopausal and perimenopausal females are protected from angiotensin II (Ang II)‐induced hypertension, whereas postmenopausal females are susceptible and respond to Ang II with a significant increase in blood pressure. Estrogen treatment during the transition into menopause prevents postmenopausal Ang II‐induced hypertension. , However, the underlying protective mechanisms of estrogen against hypertension development remain unclear.

Inflammation plays an essential role in the genesis and maintenance of hypertension. , Genetic ablation of the recombination activating gene‐1 (Rag‐1), which results in the absence of mature T and B cells, mitigates Ang II and salt‐induced hypertension in males. , The adoptive transfer of T cells into Rag‐1−/− male mice restores the magnitude of blood pressure elevation to wild‐type levels.

However, we have demonstrated that premenopausal females are protected from this T cell‐mediated hypertension. Compared with male mice, the adoptive transfer of T cells into premenopausal Rag‐1−/− mice results in attenuated blood pressure, renal T cell infiltration, and renal T cell‐dependent inflammatory cytokine expression. In contrast, the adoptive transfer of T cells into estrogen‐deplete, menopausal Rag‐1−/− mice causes a significant increase in Ang II‐induced blood pressure accompanied by an increase in renal T cell‐dependent inflammatory cytokine expression and a reduced renal T regulatory cell (Treg) population. Thus, females are protected from T cell‐mediated hypertension until the onset of menopause. Moreover, we have demonstrated that female protection is dependent on the anti‐inflammatory CD4+ Treg population.

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. Splenic T cell numbers do not increase or decrease with Ang II infusion studies, unlike those in the kidney, therefore splenic populations were chosen for this analysis of Ang II activation of signaling, in the presence and absence of estrogen. Using quantitative proteomic and phosphoproteomic approaches, we identified and profiled alterations in CD4+ T cell protein abundance and phosphorylation associated with menopausal susceptibility to Ang II hypertension.

Methods

The authors declare that all supporting data are available within the article and its online supplementary files (Data S1). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD021233 and 10.6019/PXD021233.

Experimental Animals

Eight‐week‐old, female C57BL/6J mice were purchased from the Jackson Laboratory. Mice were housed in the University Animal Care Keating/BIO5 Clean Animal Facility in standard cages, given access to water and standard mouse chow (0.25% NaCl; Harlan Teklan, 7013) ad libitum, and maintained on 12/12‐hour light/dark cycles. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Arizona.

To induce ovarian failure (menopause), a subset (n=4) of female mice received daily intraperitoneal injections of VCD (160 mg/kg i.p., Sigma V3630) for 20 consecutive days. Control (n=4) and Ang II (n=4) mice received intraperitoneal injections of vehicle (sesame oil). Vaginal cytology was measured daily to determine the onset of menopause, defined as 10 consecutive days of diestrus. VCD treated mice demonstrated confirmed ovarian failure 53±2 days from the first day of VCD injections.

Ang was infused into both premenopausal and postmenopausal mice at 800 ng/kg per minute for 14 days (Ang II and Meno/Ang II) via subcutaneous implantation of osmotic minipumps (Alzet, model 1004). The goal of the study was to identify T cell changes associated with progression into hypertension susceptibility in the VCD model of menopause (ie premenopause and postmenopausal mice +/− Ang II). We have previously shown that VCD treated mice do not have an increase in blood pressure, in the absence of Ang II, and therefore did not include for proteomic analyses. Blood pressure responses for the current study are shown in Figure S1.

Splenic CD4+ T Cell Isolation for Protein Collection

Upon euthanasia, spleens were isolated and mechanically disrupted through a 70 µm Nylon tissue separation filter (Fisher Scientific, cat. # 07‐201‐431) to create a single‐ cell suspension in 2% FBS in PBS. Splenic CD4+ cells were isolated via negative immunomagnetic separation using the EasySep™ Mouse CD4+ T Cell Isolation Kit (StemCell Technologies, cat. # 19852) according to the manufacturer’s protocol. Purity for CD4+ cells was assessed via flow cytometry by staining with surface antibodies for CD4 overnight as previously described. All samples were >88% pure and average sample purity was not different between groups (P=0.34).

Isolated CD4+ T‐cells were incubated in lysis buffer (50 mmol/L HEPES pH 7.6, 1% Triton X‐100, 50 mmol/L sodium chloride, 20 mmol/L sodium pyrophosphate, 20 mmol/L B‐ glycerophosphate disodium salt hydrate, 10 mmol/L sodium fluoride, 1.0 mmol/L sodium orthovanadate, 1.0 mmol/L phenylmethylsulfonyl fluoride, 0.017 mg/mL aprotinin, 0.01 mg/mL leupeptin) on ice for 20 minutes followed by centrifugation (18 626g, 20 minutes, 4°C). The supernatant was collected, and protein concentration was quantified using the Pierce™ BCA Protein Assay Kit.

Gene Ontology Overrepresentation and Transcription Factor Identification

For 2 pairwise comparisons (Control versus Ang II and Ang II versus Meno/Ang II), the differentially expressed proteins and phosphosites were separated into 2 groups of expression: upregulated or downregulated. The protein/phosphorylated protein groups were entered into the PANTHER Classification System (Thomas Lab at the University of Southern California, version 14.0) to identify overrepresented molecular functions and biological processes.

Differentially expressed proteins were also subject to transcription factor binding site motif analysis using Pscan (version 1.5). The default promoter region of −450 to +50 was used to identify transcriptional regulators of the differentially expressed proteins.

Phosphorylation Motif Determination and Kinase Prediction

Kinase motif analysis was conducted by input of significantly downregulated or upregulated phosphosites ±6 amino acids into iceLogo, which determines significantly overrepresented or underrepresented amino acids in a site‐specific manner within the experimental peptide sequences. Scansite 4.0 was used to predict the kinase(s) specific to each significantly downregulated or upregulated phosphosite.

Volcano Plot Visualization

Volcano plot visualization was used to identify major protein abundance and phosphorylation changes between Control and Ang II, and Ang II and Meno/Ang II samples, as defined by a log2 fold change > |1| and P≤0.05. A pseudo‐count of 1 was added to all protein abundance and phosphosite intensities to avoid a logarithm of zero. From this reduced list of proteins or phosphosites, identified from the volcano plot proteins, Euclidean distance from the origin (log2 fold change, −log10 P value from the origin [0,0]) was used to further identify an arbitrary number (4) of proteins of interest for further analysis into their functional role in T cells. These proteins had a large fold change between groups and high statical significance between groups. All figures were generated using the statistical software R (v3.3.3) (R Core Team [2017]. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R‐project.org/).

Data Analysis and Statistics

All statistical analyses presented were performed using GraphPad Prism software, the Perseus software platform, , the PANTHER Classification System, or Pscan. A Student t test was used for analyses conducted between 2 groups and one‐way ANOVA was performed in instances when all 3 experimental groups were compared. A P<0.05 with no multiple testing correction was considered significant.

Results

Ang II and Menopause Exert Distinct Effects on CD4+ T‐Cell Protein Profiles

Premenopausal females are protected against T cell‐mediated Ang II hypertension, whereas menopausal females are susceptible to T cell‐mediated Ang II hypertension. , Anti‐inflammatory CD4+FOXP3+ Tregs are part of the protective mechanism for blood pressure control in premenopausal females. To determine CD4+ T cell molecular changes associated with premenopausal resistance and menopausal susceptibility to Ang II‐induced hypertension, we subjected splenic CD4+ T cells from premenopausal mice (Control and Ang II) and menopausal mice (Meno/Ang II) to proteomic quantification (Figure 1A). Of the 7085 proteins identified, 546 were significantly different among the 3 groups (Figure 1B).

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.

Hierarchical clustering of the 546 proteins confirmed distinct CD4+ T cell proteomic differences amongst groups. The clustering results from the pattern in changes of protein abundance, which is unbiased. Ang II and Meno/Ang II groups clustered within the same dendrogram node (Figure 1C). A total of 3 principal patterns of expression were observed. Cluster 1: When compared with Control, Ang II infusion induced a stepwise increase in the expression of 267 proteins that were further induced when menopause was superimposed. Cluster 2: Conversely, Ang II infusion resulted in a stepwise decrease in the expression of 228 proteins that were further reduced with menopause. Cluster 3: The remaining 51 proteins decreased in expression in Ang II CD4+ T cells compared with Control, and this effect was reversed in menopause (Figure 1C). Pairwise comparisons revealed the largest percent of differentially expressed protein abundance changes were between Control and Meno/Ang II CD4+ T cells (41%; Figure 1D).

In Premenopausal T Cells, Ang II Increases Abundance of Proteins Associated With Transcription

To identify CD4+ T cell protein abundance changes associated with premenopausal resistance to T cell‐mediated Ang II hypertension, we analyzed the 384 differentially expressed proteins between Control and Ang II. The majority (263, 68%) were upregulated in Ang II CD4+ T cells (Figure 2A; Table S1).

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.

To provide cellular context, differentially expressed proteins were analyzed in PANTHER. Proteins upregulated in Ang II CD4+ T cells associated with biological processes involved in DNA replication and transcription (Figure 2B; Table S2) and molecular functions associated with histone modification and RNA polymerase binding (Figure 2C; Table S3). A smaller proportion of proteins were downregulated in Ang II CD4+ T cells and these associated with biological processes related to metabolic activity, including cellular respiration (Figure 2B; Table S4).

From the volcano plot, BRNP3, DAXX, CDK12, and SENP8 were the highest upregulated Ang II proteins (Figure 2D). These proteins are collectively involved in transcription regulation and ubiquitination‐like processes. , , Proteins downregulated by Ang II infusion included MPEG1, OXLA, CATR, and SERC1, and are known to be involved in regulating innate and adaptive immune function. ,

The promoter regions (−450 to +50) were analyzed by the Pscan Web Interface to identify transcription factors predicted to transcriptionally regulate differentially expressed proteins. The ZBTB family of transcription factors, which have a role in T cell lineage commitment and the formation of memory T cells, were amongst the transcription factors associated with upregulated proteins from Ang II CD4+ T cells (Figure 2E; Table S5). ZBTB7A maintains the size and function of peripheral Treg pools. The ELF family of transcription factors, that control proliferation and homing of T cells, were amongst the transcription factors associated with downregulated proteins from Ang II CD4+ T cells (Figure 2E; Table S6).

In Menopausal T Cells, Ang II Differentially Regulates KLF14 Transcription

Premenopausal females are resistant to blood pressure changes following Ang II infusion. In contrast, using the VCD model, we have shown that menopausal females respond to Ang II with a significant increase in blood pressure. To identify T cell proteins associated with the divergent blood pressure phenotype, we directly compared CD4+ T cell protein abundance from premenopausal and menopausal Ang II infused females (Ang II versus Meno/Ang II). Of the 285 differentially expressed proteins identified between the 2 groups, the majority (189, 66%) were downregulated in Meno/Ang II CD4+ T cells (Figure 3A; Table S7).

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.

Via PANTHER we classified the biological processes and molecular functions represented by differentially expressed proteins in Meno/Ang II CD4+ T cells. Proteins upregulated in Meno/Ang II CD4+ T cells were enriched in metabolic processes (Figure 3B; Table S8) and functions associated with binding and metallochaperone activity (Figure 3C; Table S9). Downregulated proteins associated with processes involved in immune responses, in particular innate T cell processes (Figure 3B; Table S10) associated with functions related to viral RNA binding (Figure 3C; Table S11).

Volcano plot visualization identified MYCN, OASL2, GPAA1, and COR2A as proteins with the greatest downregulation in Meno/Ang II CD4+ T cells compared with Ang II alone (Figure 3D). OASL2 is involved in anti‐viral responses, and COR2A regulates TLR4 induced transcription. The proteins with the greatest upregulation in Meno/Ang II CD4+ T cells were COX17, OXLA, RRP36, and EZH1 (Figure 3D). Of interest, OXLA and EZH1 regulate CD4+ T cell differentiation, proliferation, and cytokine release. , Overall the results indicate that menopause amplifies the T cell mediated inflammatory response to Ang II.

Pscan analysis of promoter binding motifs within the 189 downregulated proteins in Meno/Ang II CD4+ T cells showed transcriptional regulation by HINFP, TFDP1, KLF14, and SP4 (Figure 3E; Table S12). Promoter binding motifs within the 96 upregulated proteins specify KLF14, GABPA, GMEB1, and ZBTB7A transcriptional activity (Figure 3E; Table S13). The presence of KLF14 binding motifs in Meno/Ang II CD4+ T cell differentially expressed proteins is particularly interesting considering the inverse association of KLF14 expression with Treg function. Overall, the proteomic evaluation reveals that Ang II superimposed on menopause amplifies inflammatory pathways while potentially suppressing T regulatory responses.

Phosphoproteome Analysis of CD4+ T Cells Uncovers Distinct Effects of Both Ang II and Menopause

Using peptide residue modification readouts, we quantified the CD4+ T cell phosphoproteome from premenopausal mice (Control) and Ang II treated premenopausal (Ang II) and menopausal mice (Meno/Ang II). Mass spectrometry enabled the detection of 6059 phosphorylated sites (phosphosites), of which 466 were differentially phosphorylated among the 3 groups (Figure 4A).

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.

Distinct treatment effect was confirmed by hierarchical clustering (Figure 4B). Similar to protein abundance, clustering of Ang II and Meno/Ang II samples within the same dendrogram node was noted (Figure 4B).

Three principal patterns of peptide phosphorylation were observed across the 3 groups (Figure 4B). Pattern 1: The majority of phosphosites (248, 53%) increased from Control to Ang II to Meno/Ang II such that maximal phosphorylation was seen in Meno/Ang II CD4+ T cells. Pattern 2: 189 phosphosites (41%) linearly decreased from Control, to Ang II to Meno/Ang II. Pattern 3: 29 phosphosites (6%) increased in Ang II compared with both Control and Meno/Ang II groups. By pairwise comparisons, the majority of phosphorylation differences (43%) were uniquely different between Control and Meno/Ang II CD4+ T cells (Figure 4C).

Ang II Increases Proline‐Directed Protein Phosphorylation Linked to Chromatin Regulation by Methylation

To identify signaling cascades involved in premenopausal protection from T cell‐mediated Ang II hypertension, we analyzed the 226 differentially regulated phosphosites between Control and Ang II CD4+ T cells. Peptide phosphosites were normalized to protein abundance to account for the positive relationship between protein abundance and peptide modification frequency. Ang II increased phosphorylation at 200 phosphosites and decreased phosphorylation at 26 phosphosites (Figure 5A; Table S14). Classification of proteins associated with differentially regulated phosphosites between Control and Ang II was performed with PANTHER. Proteins with upregulated phosphorylation were enriched for histone methylation, chromatin regulation, enzyme activity, and DNA binding (Figure 5B and 5C; Tables S15 and S16). No known annotated biological processes were enriched within the 26 downregulated phosphosites.

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.

Phosphosites with greatest phosphorylation upregulation in Ang II CD4+ T cells included FBRL S130, IWS1 S666, PININ S346, and DAXX S515 (Figure 5D). Of note, DAXX decreases TCR‐induced proliferation and is a critical regulator of T cell homeostasis. Conversely, Ang II resulted in the downregulation of 26 phosphosites, of which BNIP2 S114, HNRPF Y276, HS90A S252, and HCLS1 S333 were phosphorylated the least compared with Control CD4+ T cells. Overexpression of HNRPF has been shown to reduce Treg suppressive function in vitro, through direct interaction with FOXP3. Whereas, upon T cell activation, HCLS1 interacts with actin at immunological synapses and promotes chemokine induced T cell migration. ,

Differentially regulated phosphosites (±6 amino acids) were uploaded into iceLogo to determine putative phosphorylation motifs within modified proteins. In downregulated phosphosites (Figure 5E), serine and tyrosine were significantly overrepresented as the site of modification. Moreover, the phosphorylation motif (Figure 5E) indicated inactivity of proline directed kinases, as well as some acidophilic (residues D/E) and basophilic kinases (K). Scansite 4.0 was used to determine specific kinases associated with each downregulated phosphosite. Scansite identified kinases included CK2, CDK1, and CDK5, which were predicted to interact with amino acid motifs surrounding 9 of the 26 downregulated phosphosites (Figure 5E). It is important to note that increased phosphatase activity, rather than reduced kinase activity likely explains the decreased phosphorylation.

This analysis was repeated for upregulated phosphosites where serine and threonine residues were found to be overrepresented at the site of modification (Figure 5F). Additionally, the phosphorylation motif suggested increased activity of proline directed and acidophilic kinases, and the possibility of phosphorylation induced phosphorylation. Kinases predicted to have increased activity in Ang II CD4+ T cells include CDK1, CDK5, ERK1, PKA, and GSK3.

Meno/Ang II Results in Decreased Proline‐Directed Protein Phosphorylation, Notably S405 Within TLN1, a Positive Regulator of Treg Maintenance

To identify phosphorylation activity and protein signaling associated with menopausal susceptibility to Ang II hypertension, we focused on differentially expressed phosphosites between Ang II and Meno/Ang II CD4+ T cells. After peptide phosphorylation normalization to protein abundance, 115 of 207 phosphosites (56%) were downregulated in Meno/Ang II CD4+ T cells (Figure 6A; Table S17). As a collective group, Meno/Ang II CD4+ T cell proteins with significantly upregulated phosphorylation associated with one molecular function, actin binding (Figure 6C; Table S18). Whereas, proteins with significantly downregulated phosphorylation associated with protein localization to the nucleus, translation initiation, and nucleic acid binding (Figure 6B and 6C; Tables S19 and S20).

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.

Downregulated phosphosites included EIF3B S120, MYCN S350, TOE1 S349, and TLN1 S405 (Figure 6D). Of interest, TLN1 is necessary for the maintenance and survival of Tregs through positive regulation of Treg IL‐2Rα (CD25) expression. Further, 92 phosphosites were upregulated in Meno/Ang II CD4+ T cells. Phosphosites with the greatest phosphorylation upregulation in Meno/Ang II CD4+ T cells included PRP4B S278, MP2K2 S23, PHAR4 S118, CP100 S244 (Figure 6D). Of note, MP2K2 stimulates ERK1/2 activity, which influences the polarization of CD4+ T cells to a Th17 lineage.

Downregulated and upregulated phosphosites (±6 amino acids) in Meno/Ang II CD4+ T cells were uploaded into iceLogo. Decreased phosphosites predominantly consisted of a modified serine with neighboring acidophilic amino acid residues (Figure 6E). Analysis of kinase specificity for this motif suggested that CK2, CDK1, DNA PK, GSK3, and PKA had decreased activity at the identified phosphosites in Meno/Ang II CD4+ T cells compared with Ang II CD4+ T cells (Figure 6E). Motif analysis of upregulated phosphosites identified an overrepresentation of proline directed serine phosphorylation in Meno/Ang II CD4+ T cells (Figure 6F). Kinases responsible for increased phosphorylation at the identified sites were predicted to be ERK1, CDK1, CDK5, GSK3, and AKT (Figure 6F).

Discussion

The goal of this study was to identify molecular targets responsible for the loss of female protection against Ang II‐induced T cell‐mediated hypertension following menopause. The major findings of this study were: (1) Ang II infusion in both premenopausal and menopausal mice increased CD4+ T cell protein abundance and phosphorylation associated with DNA and histone methylation; (2) in menopausal T cells, Ang II infusion increased phosphorylation of MP2K2, an upstream regulator of ERK; and (3) in menopausal T cells, Ang II infusion decreased phosphorylation of TLN1, a key regulator of IL‐2Rα and FOXP3 expression. These findings identify novel T cell pathways that suggest increased T cell‐mediated inflammation during postmenopausal hypertension (Figure 7).

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.

Several studies support the role of epigenomic regulation in the development of hypertension. A well characterized epigenetic modification known to influence hypertension development is DNA methylation, which within the gene promoter, is generally associated with gene repression. , In the Dahl salt‐sensitive rat, increased blood pressure caused by a high salt (HS) diet is associated with increased DNA methylation in the renal outer medulla and in renal T cells. Inhibition of HS‐induced methylation and demethylation within the renal outer medulla significantly reduced MAP by HS day 7. Global inhibition of DNA methyltransferase activity in non‐terminally differentiated cells via decitabine administration significantly blunted HS‐induced MAP and proteinuria. Together these studies suggest a causal contribution of DNA methylation and demethylation in hypertension development.

Another epigenetic modification known to be associated with hypertension is histone methylation. Histone 3 trimethylation of the fourth lysine (H3K4me3) is a hallmark of active DNA transcription whereas histone 3 trimethylation of the twenty‐seventh lysine (H3K27me3) and trimethylation of the ninth lysine (H3K9me3) are associated with transcription repression. , Spontaneously hypertensive rats (SHRs) have been shown to express increased recruitment of H3K4me3 to the ACE promoter within aortic, heart, and kidney tissue, that corresponded to local increased ACE expression. Valsartan treatment of SHRs significantly reduced enrichment of H3K4me3 to the ACE promoter in heart and kidney tissue, reduced ACE expression in heart and kidney, and reduced blood pressure.

The current study found that in premenopausal mice, Ang II infusion increased CD4+ T cell protein abundance and phosphorylation associated with the following processes/functions: DNA methylation, H3K4me3, H3K27me3, negative regulation of chromatin silencing, and histone methyltransferase activity. However, as seen in Figures 2A and 5A proteins with increased abundance and phosphorylation in Ang II compared with Control were similarly upregulated in Meno/Ang II, suggesting that similar to other hypertension models, Ang II stimulates DNA and histone methylation.

When protein abundance changes between Meno/Ang II and Ang II were analyzed, differentially expressed proteins consistent with increased KLF14 transcription factor activity were identified. KLF14 expression is inversely associated with FOXP3 expression and is thought to repress FOXP3 transcription via recruitment of repressive H3K9me3 to the FOXP3 promoter. Furthermore, protein abundance of EZH1, a methyltransferase, was increased in Meno/Ang II CD4+ T cells. EZH1, along with EZH2, is associated with the repressive H3K27me3, and is known to silence IFNγ, GATA3, and IL‐10 loci in naïve CD4+ T cells. As seen in Figure 7, the increase in KLF14 activity and EZH1 expression are 2 pathways that may contribute to the susceptibility of menopausal mice to T cell‐mediated hypertension.

In T cells sustained ERK activation, downstream of TCR signaling, is known to correlate with T cell differentiation and proliferation, whereas transient ERK activation correlates with T cell unresponsiveness and apoptosis. Activation of ERK via phosphorylation mediates the degradation of NF‐κB inhibitory protein IkBα, leading to NF‐κB nuclear translocation and increased pro‐inflammatory gene transcription, specifically mediating Th17 cell development. In splenocytes isolated from asthmatic mice, in vitro inhibition of ERK activation, via PD98059, reduced secretion of pro‐inflammatory Th17 associated cytokine, IL‐17, and increased secretion of Treg associated cytokine, IL‐10. Furthermore, treatment of asthmatic mice with a voltage gated potassium channel antagonist reduced lung expression of phosphorylated ERK and nuclear NF‐κB, reduced serum and bronchoalveolar lavage fluid IL‐17 concentration and reduced the splenic percentage of CD4+IL17A+ T cells. This reduction in Th17 associated response was accompanied by increased serum and bronchoalveolar lavage fluid IL‐10 concentration and increased splenic percentage of CD4+FOXP3+CD25+ T cells.

Further evidence supports ERK activation as a mediator of Th17 cell development. Inhibition of ERK activation in isolated murine CD4+ T cells cultured under Th17 polarization conditions led to less IL‐17 secretion, but more IL‐10 and TGFβ. Additionally, ERK inhibitor treated T cells under Th17 polarization conditions suppressed naïve T cell proliferation at levels comparable with that of purified Tregs. Moreover, adoptive transfer of ERK inhibitor treated T cells, after 5 days under Th17 polarization culture, into Rag‐1−/− mice, resulted in reduced colitis pathological scores as compared with transfer of unpolarized and Th17 polarized T cells.

The current study found that compared with premenopausal mice, Ang II infusion in menopausal mice increased phosphorylation of phosphosites associated with ERK activity, increased phosphorylation of MP2K2 (also known as MEK2), and increased CD4+ T cell protein abundance of OXLA (Figure 7). Of the 92 identified phosphosites with increased phosphorylation in T cells from Meno/Ang II treated females, 35% were predicted to be targeted by ERK. Increased MP2K2 activity is consistent with the increased predicted ERK activity at proline‐directed phosphosites in Meno/Ang II. Furthermore, OXLA expression is dependent on retinoic acid orphan receptor (RORγt) expression, the main transcription factor associated with Th17 cell development. , Consistent with this, OXLA expression is upregulated in Th17 cells. As seen in Figure 7 increased ERK activation and increased Th17 differentiation in menopausal CD4+ T cells is a pathway to consider in the development of menopausal hypertension.

In addition to a role for Th17 cells in hypertension development , , several studies support a role for Tregs in hypertension prevention. , , Recently TLN1, a cytoskeletal protein essential in mediating integrin activation, has been shown to be essential for Treg proliferation and function. CD4+ T‐cell specific TLN1 deletion in TLN1fl/flCD4Cre mice resulted in spontaneous lymphocyte activation. This spontaneous lymphocyte activation was accompanied by a depletion of splenic CD4+FOXP3+ Tregs. Further, splenic CD4+FOXP3+ Tregs from TLN1fl/flCD4Cre mice have decreased p‐STAT5, IL‐10, and IL‐2Rα expression and decreased FOXP3 median fluorescence intensity on a per cell basis. This suggests an important role for TLN1 in regulating Treg survival through maintenance of increased IL‐2Rα expression (also known as CD25) and downstream p‐STAT5 signaling. STAT5 phosphorylation occurs directly downstream of the IL‐2R and is known to block Th17 cell development and promote Treg differentiation and maintenance of function by targeting the FOXP3 promoter.

The importance of TLN1 in Treg function was further confirmed in Treg specific TLN1 depleted (TLN1fl/flFOXP3Cre) hemizygous mice. TLN1fl/flFOXP3Cre hemizygous mice developed systemic autoimmunity that ultimately resulted in death by 2 to 3 months of age. These mice exhibited increased splenic percentages of activated (CD62LloCD44hi) CD4+ and CD8+ cells that were highly proliferative and expressed increased levels of IFNγ and TNFα. This increase in inflammation was accompanied by reduced IL‐2Rα and FOXP3 expression in TLN1 deficient Tregs, again suggesting an important role for TLN1 in mediating Treg suppressive function.

The current study found that compared with premenopausal mice, Ang II infusion in menopausal mice decreased CD4+ T cell phosphorylation of TLN1 at S405, however little is known about the role of phosphorylation at S405 in TLN1 function. TLN1 phosphorylation by CDK5 at S425 has been shown to activate β1 integrins in metastatic prostate cancer cells. Given the proximity of our identified phosphosite to a known activating phosphosite and the major integrin binding domain of TLN1 (AAs 86–403), phosphorylation at S405 may regulate TLN1‐integrin binding. Therefore, decreased phosphorylation of TLN1 at S405 and downstream IL‐2R signaling in CD4+ T cells may contribute to reduced Treg function and increased hypertension susceptibility in menopausal females (Figure 7).

In conclusion, this proteomic study of T cells from premenopausal and postmenopausal females identifies Ang II regulated T cell pathways. In postmenopausal females, Ang II signaling and an increase in blood pressure potentially regulate the T cell proteome. Taken together we identify novel T cell pathways that contribute to enhanced T cell inflammation leading to hypertension in postmenopausal females.

Conclusions

The present study identifies T cell function and signaling during the progression of hypertension across the postmenopausal transition. CD4+ T cell proteomic and phosphoproteomic analyses revealed 285 differentially regulated proteins and 207 differentially regulated phosphorylation sites between Ang II infused menopausal and premenopausal mice. Pathways including ERK, TLN1, EZH1, and KLF14 were identified as potential regulators of CD4+ T cell function during menopausal hypertension. These data identify novel T cell pathways that are dysregulated in postmenopausal females, contributing to greater T cell‐mediated inflammation and an increased incidence and severity of hypertension. Selective manipulation of the identified T cell molecular targets may prove to be an effective strategy to inhibit the inflammation and renal damage associated with severe postmenopausal hypertension.

Sources of Funding

This work was funded by grants from the National Heart, Lung, and Blood Institute, RO1HL131834, T32HL007249 and National Institute of Diabetes and Digestive and Kidney Diseases RO1DK098493.

Disclosures

None.

Data S1

Tables S1–S20

Figure S1

References ,

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