Gene Silencing and Haploinsufficiency of Csk Increase Blood Pressure

Objective Recent genome-wide association studies have identified 33 human genetic loci that influence blood pressure. The 15q24 locus is one such locus that has been confirmed in Asians and Europeans. There are 21 genes in the locus within a 1-Mb boundary, but a functional link of these genes to blood pressure has not been reported. We aimed to identify a causative gene for blood pressure change in the 15q24 locus. Methods and Results CSK and ULK3 were selected as candidate genes based on eQTL analysis studies that showed the association between gene transcript levels and the lead SNP (rs1378942). Injection of siRNAs for mouse homologs Csk, Ulk3, and Cyp1a2 (negative control) showed reduced target gene mRNA levels in vivo. However, Csk siRNA only increased blood pressure while Ulk3 and Cyp1a2 siRNA did not change it. Further, blood pressure in Csk+/- heterozygotes was higher than in wild-type, consistent with what we observed in Csk siRNA-injected mice. We confirmed that haploinsufficiency of Csk increased the active form of Src in Csk+/- mice aorta. We also showed that inhibition of Src by PP2, a Src inhibitor decreased high blood pressure in Csk+/- mice and the active Src in Csk+/- mice aorta and in Csk knock-down vascular smooth muscle cells, suggesting blood pressure regulation by Csk through Src. Conclusions Our study demonstrates that Csk is a causative gene in the 15q24 locus and regulates blood pressure through Src, and these findings provide a novel therapeutic target for the treatment of hypertension.


Introduction
Blood pressure is influenced by a variety of mechanisms that involve many genetic factors. To detect genetic markers for blood pressure, genome-wide association studies (GWASs) have been performed using large human samples from various ethnic groups and have identified many genetic loci that are associated with blood pressure and hypertension. The Korean Association REsource (KARE) [1], the Global Blood Pressure Genetics (GlobalBPgen) [2], Cohorts for Heart and Aging Research in Genome Epidemiology (CHARGE) [3], the Asian Genetics Epidemiology Network Blood Pressure (AGEN-BP) [4], and International Consortium for Blood Pressure (ICBP) [5] have conducted GWASs on blood pressure and hypertension, identifying 33 independent loci that have reached a genome-wide significance level.
ULK3, serine/threonine kinase, mediates Hedgehog signaling during embryonic development and has been implicated in the maintenance of tissue homeostasis and neurogenesis in adults [17]. During the cell cycle, phosphorylation of Ulk3 is important in the G2/M phase, suggesting a role in cell cycle progression [18].
There has been no functional study to support the link of these genes to blood pressure. Based on eQTL analysis studies, mouse homologs Csk, Ulk3, and Cyp1a2 (negative control) were examined for blood pressure change in siRNA-injected mice after testing for in vivo silencing by siRNAs injection. We showed that only Csk siRNA injection increased blood pressure while Ulk3 and Cyp1a2 siRNA injections did not change it. Our results along with the eQTL analysis indicate that CSK is a causative gene in the 15q24 locus. We also confirmed that haploinsufficiency of Csk increased blood pressure in Csk +/heterozygote mice compared to the wild-type. Further, inhibition of Src, a Csk target ameliorated hypertension in Csk +/heterozygote mice, suggesting that Csk regulates blood pressure through Src.

Animal research and ethics statement
All mice were housed and handled in a pathogen-free facility of College of Pharmacy at Kyung Hee University in compliance with the Guide for the Care and Use of Laboratory Animals, fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. The mice were maintained on a 12-hour light/dark cycle at a constant temperature with free access to food (LabDiet 5L79, St. Louis, MO, USA) and water. Every effort was made to minimize the number of sacrificed animals and their suffering. Animals were anesthetized by intraperitoneal (i.p.) injection of tribromoethanol (18 ml of the working solution per kg body weight), of which the working solution was diluted 40-fold in 0.9% NaCl from the stock solution (10 g 2,2,2, tribromoethanol dissolved in 10 ml tertiary amyl alcohol) and euthanized by removing the heart. The experiment was approved by the local committee for the Care and Use of Laboratory Animals, College of Pharmacy Institutional Animal Care and Use Committee (license number: KHP-2010-04-06).

The in vivo delivery of siRNA
The selection and in vivo delivery of siRNA have been previously described [19][20][21]. In brief, more than 3 siRNAs per gene (Csk, Ulk3, and Cyp1a2) were synthesized by Genolution (Seoul, Korea), and only one of them was selected for in vivo injection. To determine their silencing efficacy, 20 nM of the siRNAs was transfected into B16F10 cells and NIH3T3 cells using G-Fectin (Genolution, Seoul, Korea) and Lipofectamine 2000 (Invitrogen, Carlsbad, CA) respectively according to their manufacturer's instructions. TRP53 siRNA was used as a positive control for all in vitro transfection experiments. The target siRNAs and scrambled control siRNA sequences are shown in S4 Table. For the in vivo delivery into mice, polyethylenimine called as in vivo-jetPEI™ (Polyplus, 201-10G, Illkirch-Graffenstaden, France) was used as the transfection reagent. According to the manufacturer's instruction, 50 μg of siRNA and 6.5 μl of in vivo-jetPEI (N/P charge ratio of 6) were diluted with 50 μl of 10% glucose solution and 50 μl of sterile H 2 O. The solution was vortexed gently and left for 15 min at room temperature. The mixture was injected into the tail veins of 7-9-week-old, female BALB/c mice, and 24 hours after the last injection, the treated mice were used for experiments (i.e., blood pressure measurement, collection of tissues for mRNA quantitation or for Western blotting analysis). The 1-, 2-or 3-time injection means once per day for 1, 2, or 3 days.

Quantitative real-time PCR
Total RNA was extracted from the tissues of siRNA-injected mice 24 hours after the last injection using TRIzol (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from 500 ng of total RNA using the PrimeScript™ RT kit (TaKaRa, Shiga, Japan) according to the manufacturer's protocol. Quantitative real-time PCR analysis was performed using SYBR Green I (TaKaRa, Shiga, Japan) on the ABI Step One Real-Time PCR system (Applied Biosystems, Foster, CA) using the following program: 45 cycles at 95°C for 10 s, 60°C for 15 s, and 72°C for 20 s. The primer sequences are shown in S5 Table. To present the data as the fold-change of relative expression of "case" (e.g. target siRNAtreated, Csk +/-) over "control" (e.g. control siRNA-treated, Csk +/+ ), the 2 -ΔΔCt method was used as previously described [22]. First, the Gapdh Ct value was subtracted from each target gene Ct value to obtain the delta Ct (ΔCt) value for normalization and the ΔCt of the "control" sample was averaged. Second, the averaged ΔCt value of the "control" sample was subtracted from each ΔCt value of the "case" sample to obtain the delta delta Ct (ΔΔCt = ΔCt case-ΔCt control). Third, the ΔΔCt values are converted to the linear form using the term 2 -ΔΔCt . The Standard Error of the Mean (SEM) is calculated from the final 2 -ΔΔCt values. Statistical significance was calculated for differences between the 2 -ΔCt values of the "control" and those of the "case" samples.

Culture of VSMC
The T/G HA-VSMC (vascular smooth muscle cell) line (CRL-1999) was purchased from ATCC (American Type Culture Collection, Virginia, USA). This line of VSMC is a normal human cell line established from the normal aorta of an 11-month old child and one of the best-characterized cellular models for the analysis of vascular smooth muscle cell biology. Cells were cultured in F-12K Medium (ATCC, No.  supplemented with 0.05 mg/ml ascorbic acid, 0.01 mg/ml bovine insulin, 0.01 mg/ml human transferrin, 10 ng/ml sodium selenite, 0.03 mg/ml Endothelial Cell Growth Supplement, 10 mM HEPES, 10 mM TES, and 10% fetal bovine serum under a 5% CO 2 in air-ventilated incubator. To knock-down Csk, VSMCs were transiently transfected with control or Csk siRNA using Lipofectamine 2000 and treated for 24 hours with DMSO or PP2 after 24 hours transfection and then proteins were extracted for Western blotting.

Western blotting
Total proteins were extracted from mouse tissues using PRO-PREP protein extraction solution (Intron Biotechnology, Gyeonggi-Do, Korea) according to the manufacturer's instruction. Protein concentrations were measured by Bradford assay [23].

Blood pressure measurement
The protocol for blood pressure measurement has been previously described [19][20][21]. In brief, blood pressure was recorded intra-arterially with a computerized data acquisition system (AD Instruments, Bella Vista, Australia). First, mice (7-9-week old, female, 20-25 g) were anesthetized by intraperitoneal (i.p.) injection of tribromoethanol (18 ml of the working solution per kg body weight), of which the working solution was diluted 40-fold in 0.9% NaCl from the stock solution (10 g 2,2,2, tribromoethanol dissolved in 10 ml tertiary amyl alcohol). The intraarterial catheter (a polyethylene tube; 0.2 mm I.D., 0.5 mm O.D.; Natsume, Tokyo, Japan) filled with 0.9% NaCl containing 100U/ml heparin was connected to the system. Second, while on anesthetics, a small incision was made to draw out the right carotid artery of the mouse. The catheter was inserted into the right carotid artery through a small puncture and tightly ligated with 4-0 silk suture thread around the artery. Third, blood pressure in the vessel was transmitted along the catheter to the transducer's diaphragm (MLT0699 Disposable BP Transducer, AD Instruments, Bella Vista, Australia). The diaphragm signal was amplified through a bridge amplifier and recorded on a Power Lab system (Lab Chart 7.2, AD Instruments, Bella Vista, Australia).
Blood pressure was monitored for 2 hours after injection of the anesthetic and presented as the average value at each minute between 40 and 80 min (a linear graph) and as the average value during 40 minutes from 40 to 80 min (a bar graph) because blood pressure during the presented period was considered enough stable as evidenced by multiple repeated experiments.
To test the effect of PP2 (Merck Millipore, 529573, Darmstadt, Germany) or PP3, a negative control inhibitor (Merck Millipore, 529574, Darmstadt, Germany), blood pressure was measured in Csk +/and Csk +/+ mice 24 hours after i.p. injection of PP2 or PP3 at doses of 2 or 10 μg per kg body weight.

Statistical analysis
Statistical analysis was performed using SPSS (PASW Statistics 22.0), and all data between case and control groups were analyzed by Mann-Whitney U-test since the U-test was in general considered more powerful than the t-test [24]. All data were expressed as mean ± SEM. P < 0.05 was considered statistically significant. Ã P<0.05, ÃÃ P<0.01 versus respective controls.

Results
Injection of Csk, Ulk3, and Cyp1a2 siRNA reduces their respective target mRNA levels in mouse tissues Fig 1A shows the locations of nearby genes including CYP1A2, CSK, and ULK3 on the human 15q24 locus with the comparable gene locations on mouse chromosome 9 in the opposite direction. The lead SNP (rs1378942 designated as Ã ) is located in the first intron of CSK. To test the effect of candidate gene variation on blood pressure, we used silencing of candidate genes by siRNA injection into mice in vivo. Both mouse homologs Csk and Ulk3 were selected as comparably strong candidate genes among nearby genes on the 15q24 locus based on eQTL analysis studies (S1 Table). Cyp1a2 was tested as a negative control because there has been reported neither significant association with blood pressure in GWAS or significant link to the lead SNP (rs1378942) in eQTL resources (S1 and S3 Tables). Regarding its functional role, CYP1A2 is associated with habitual caffeine consumption, and mediates drug metabolism and the synthesis of cholesterol, steroids, and lipids (S3 Table) [25,26].
Three siRNAs per gene were synthesized and tested for their silencing efficacies in NIH3T3 and B16F10 cells, and the most effective siRNA was selected for in vivo injection (S4 Table). The selected siRNAs caused Csk, Ulk3, and Cyp1a2 levels to decline by 40.1%, 74.5%, and 44.9% in cells, respectively. The siRNAs were mixed with polyethylenimine and injected into mouse tail veins. Then, to estimate the systemic change by siRNA injection, we measured target genes mRNA levels 24 hours later by quantitative real-time PCR, comparing them between candidate gene siRNA-and control siRNA-injected mouse tissues. After 1-time injection, Csk mRNA levels decreased significantly in heart by 37% (P = 0.038), in kidney by 20% (P = 0.038), and in lung by 29% (P = 0.017), respectively, and Ulk3 mRNA levels fell in lung by 32% (P = 0.001) (Fig 1B and 1C). Cyp1a2 mRNA levels fell in liver by 49% (P = 0.036) and in kidney 21% (P = 0.032) (Fig 1D).

Csk siRNA injection increases blood pressure
To study the functional effect of siRNA injection, blood pressure was measured 24 hours after the last injection of Csk, Ulk3, or Cyp1a2 (negative control) siRNAs.
To confirm reduction of the target gene mRNA, Csk, Ulk3, and Cyp1a2 mRNA levels were measured in mice tissues after 2-time injection of each siRNA. Csk mRNA levels decreased significantly in heart by 31% (P = 0.001) and in liver by 19% (P = 0.01), respectively, and Ulk3 mRNA levels also decreased in liver by 34% (P = 0.001), in spleen by 37% (P = 0.001), and in lung by 44% (P = 0.003), respectively (Fig 2C and 2F). Cyp1a2 transcripts also fell in liver by 32% (P = 0.045) after 2-time injection (Fig 2I). The results showed that only Csk gene knock- down induced a significant change in blood pressure although all three genes (Csk, Ulk3, and Cyp1a2) were knock-downed by siRNA injections in various tissues.

Csk protein levels stay decreased in mouse tissues after siRNA injection
To test the lasting effect of siRNA silencing, Csk siRNAs were injected into mice once per day for 3 days (3-time injection). Blood pressure significantly differed between Csk siRNA-treated mice (87.4 mmHg, N = 7) and control mice (80.6 mmHg, N = 7) (P = 0.017) even after 3-time injection (Fig 3A and 3B).
To study Csk protein levels in Csk siRNA-injected mice, Western blotting for Csk was performed using mouse lung tissues. Csk protein levels declined after all 1-, 2-and 3-time injections in lung tissues (Fig 3C). After 2-time injection of Csk siRNA, Csk protein levels fell in lung by 53% (P = 0.016) (Fig 3C and 3D). These data imply that Csk protein levels may stay decreased systematically after repeated injections of siRNAs despite of rebounding mRNA levels.
Since Csk is a negative regulator of Src protein [14] and Src plays a role in contraction of vascular smooth muscle cells [27], Western blotting analysis was performed for Csk and phosphorylated Src proteins in the aorta of Csk +/and Csk +/+ mice. Csk protein levels were reduced by 52% (P = 0.002) in the Csk +/aorta (Fig 4D and 4E). While the total Src protein was not changed in Csk +/aorta, phosphorylation of Src Y416 (active) significantly increased by 38% (P = 0.046) and that of Src Y527 (inactive) decreased by 25% (P = 0.01), suggesting Src as a target of Csk in the aorta (Fig 4D and 4E).
To confirm inhibition of Src by PP2, we performed Western blotting analysis for phosphorylated Src proteins in the aorta of Csk +/and Csk +/+ mice treated with 10 μg/kg PP2. As shown in Fig 5C and 5D, phosphorylated Src Y416 (active) was significantly reduced in the PP2-treated Csk +/aorta compared to vehicle-treated aorta and phosphorylated Src Y527 (inactive) was also slightly decreased.
Further to study regulation of Src by Csk in functionally relevant cell types, we cultured human vascular smooth muscle cells (T/G HA-VSMC, ATCC), transfected cells with Csk siRNA, and then treated with 10, 25, 50 μM PP2 for 24 hours. The Western blotting analysis showed that phosphorylation of Src Y416 was conspicuously increased by Csk siRNAs transfection in VSMCs consistent with the result from the Csk +/mice aorta (Fig 5E). Moreover, PP2 treatment dose-dependently decreased phosphorylation of Src Y416 in Csk siRNA-transfected cells (Fig 5E). Phosphorylation of Src Y527 did not significantly change by Csk siRNAs transfection, but seemed to decrease by PP2 treatment in VSMCs (Fig 5E).
These results suggest that Src is specifically involved in blood pressure regulation by Csk.

Discussion
It has been a challenging task to identify a causative gene in a locus that is associated with blood pressure. Therefore, there are very few genes identified as causative so far despite of multiple candidates associated with blood pressure GWASs. For that reason, we utilized two-step approaches. First, we selected the most relevant candidate genes in a locus by using the eQTL analysis studies. Then, we tested the effect of gene knock-down on blood pressure after the in vivo delivery of candidate gene siRNA into mice. We previously reported ATP2B1 in the 12q21 locus [20] as a causative gene by testing the effect of target gene siRNA silencing on blood pressure. The recent eQTL study showed that the expression of ATP2B1 transcripts was associated with rs1401982 and rs17249754 (rs1401982, P = 1.4×10 −24 for all common SNPs; rs17249754, P = 5.5×10 −4 for 1,167 trait-associated SNPs in blood) [11], supporting our previous study using in vivo siRNA silencing. The SORT1 gene is another good example that a specific gene is identified as associated with a variant (rs599839) in the 1p13 locus by an eQTL study (P = 6.11×10 −55 in liver) and that rs629301 in LD with rs599839 is associated with plasma low-density lipoprotein cholesterol (LDL-C) in GWAS (P = 1×10 −170 ) [28,29]. Musunuru et al. (2010) has clearly demonstrated that rs12740374 in LD with rs599839 and rs629301 is a causative variant because its polymorphism alters hepatic expression of the SORT1 gene and knockdown by Sort1 siRNA in mouse liver changes plasma LDL-C levels [30]. In the present study we used the eQTL resources more aggressively to sort out prominent candidates. Through mining the eQTL resources, CSK and ULK3 were selected in the 15q24 locus for further study. Target gene silencing was successfully tested in vivo according to the protocol we have previously established. As a result, Csk in the 15q24 locus was identified as a causative gene for the blood pressure change and furthermore confirmed in a knockout mouse model. Therefore, the present study and previous ones support that in vivo siRNA silencing combined with the eQTL analysis is a very useful tool to identify a causative gene among candidate genes.
However, we observed a difference in the directionality of the effect of suppression of Csk in mice from the results obtained in human GWAS and eQTL studies that the 'C' allele of the lead SNP (rs1378942) is associated with higher blood pressure and higher CSK transcripts [2, 5-7, 11, 12]. We considered three possible reasons for the conflicting results. One possibility is a molecular and functional difference between the human CSK and mouse Csk gene. But it is very unlikely since the human CSK and mouse Csk proteins are identical except one residue at 123 within SH2 domain, not within a functional domain such as the kinase domain. The other one is a recording error for SNP alleles in eQTL databases. We personally corresponded with authors of Fehrmann et al. (2011) and Grundberg et al. (2012) papers but were provided with a solid confirmation for their databases. The third reason might be the lack of the functional tissue in eQTL resources. We speculate that the correct eQTL data may be unavailable until the functional tissue of CSK for blood pressure regulation is revealed. Despite of still unsolved conflicts, we believe that our results provide an important clue for how the decreased Csk gene activity is associated with hypertension in Spontaneously Hypertensive Rats (SHR) [16], as explained in the following section.

Csk regulates blood pressure through Src
Csk was identified as a tyrosine kinase of Src, a non-receptor tyrosine kinase [14]. Csk inactivates Src by increasing phosphorylation of Src Y527 , acting as a negative regulator of Src (Fig 6) [31]. Phosphorylation of Src Y416 is increased in the Csk +/mouse aorta and in Csk knock-down cells than in the wild-type (Fig 5C, 5D and 5E). Furthermore, inhibition of Src decreases high blood pressure in Csk +/mice (Fig 5A and 5B), strongly suggesting that hypertension occurs through Src.
Src is one member of eight Src family tyrosine kinases (c-Src, c-Yes, Fyn, c-Fgr, Lyn, Hck, Lck, and Blk) [31]. Among them, the activity of Src is important in Ang II-induced vasoconstriction of vascular smooth muscle cells (VSMCs). Ang II induces phosphorylation of Src Y416 via angiotensin type 1 receptor (AT1R) in rat VSMCs [32]. Ang II-induced intracellular Ca 2+ mobilization is blunted in Src -/-VSMC and vascular contraction is inhibited by PP2, a Src inhibitor [27]. Interestingly low activation of Csk is associated with increased Ang II-mediated Src signaling in VSMCs of Spontaneously Hypertensive Rats (SHR) [16]. These reports and our findings suggest that the Csk-Src signal may be a key regulatory point in Ang II-mediated vasoconstriction and blood pressure regulation (Fig 6).
Downstream of the Csk-Src signal, extracellular signal-regulated kinases (ERK1/2) may play an important role in vasoconstriction. In SHRs, inhibition of ERK signaling by U0126 reduces hypertension, vascular thickening, and smooth muscle myosin light chain kinase expression [33]. Moreover, Ang II-mediated ERK1/2 activation requires Src in rat VSMCs [16,34]. Therefore, we speculate that Csk negatively modulates Ang II-mediated Src/ERK signaling in vasoconstriction of VSMCs.

Src plays important roles in Ang II-mediated signaling in multiple tissues
Ang II is a major effector hormone of the renin-angiotensin system and has various effects that are mediated by AT1R to raise blood pressure, including direct vasoconstriction in the vascular smooth muscle, indirect vasoconstriction by noradrenaline release from the sympathetic nerve system, sodium reabsorption in the kidney, sodium and fluid retention by aldosterone secretion from the adrenal gland, and vascular growth stimulation [35].
In addition to already mentioned role of Src in VSMCs, Src activation is required for Ang IImediated functions in the kidney and in the brain. In the renal proximal tubule-like cells, PP2 and Src siRNA inhibits Ang II-induced epithelial-to-mesenchymal transition (EMT) associated with progressive kidney damage [36]. Also Src is activated in Ang II-induced fluid reabsorption via apical Na + /H + exchanger (NHE) and basolateral Na + -HCO 3 cotransporter (NBC) and Csk overexpression inhibits activation of both NHE and NBC [37,38]. In the distal tubule of kidney, Src is required for inhibition of the renal outer medullary potassium (ROMK) channel during hypovolemia (low blood volume) by low potassium diet [39][40][41][42]. Therefore, hypertension by low potassium diet may be associated with the inhibitory effect of Src on the ROMK channel.
In the hypothalamus of hypertensive rats induced by two-kidney one-clip (2K1C), Src activation is higher than sham-operated rats and is more increased by Ang II [43]. Two specific Src inhibitors (PP2, SU6656) abolish increase in sympathetic nerve activity and blood pressure of 2K1C rats, suggesting that Src in the brain mediates sympathetic activation by renovascular hypertension.
Considering that Src plays regulatory roles in diverse tissues, hypertension in Csk +/mice may rather be the combined result of differential effects by chronic Src activation in multiple tissues (e.g., vasculature, kidney, or/and brain) than the result of a defect in one particular tissue. Therefore, to fully understand the mechanism of hypertension in Csk +/mice, changes in Src-associated signaling need to be further studied in each tissue.
In summary, we have identified CSK a causative gene in the blood pressure GWAS locus 15q24 by selecting candidate genes using the eQTL analysis studies, by verifying the functional effect of genes using in vivo siRNA delivery in mice, and by confirming our findings in knockout heterozygote mice. Furthermore, we showed that Csk involves Src by negatively regulating its activity to modulate blood pressure. Our result that Csk regulates blood pressure through Src suggests a novel pathway for the development of hypertension and provides a therapeutic target for the treatment of high blood pressure. Supporting Information S1 Fig. No effect on blood pressure by PP3 in Csk +/+ and Csk +/mice. (A and B) Csk +/and Csk +/+ mice were i.p. injected with DMSO as control (CON) or PP3 (10 μg per kg body weight), a negative control for PP2, and blood pressure was measured 24 hours after injections. (PDF) S1