FRNK Plays A Critical Role In Inhibition Of Cardiac Hypertrophy In Vitro And In Vivo

FRNK plays a critical role in inhibition of cardiac hypertrophy in vitro and in vivoBackground & ObjectiveCardiac hypertrophy is an adaptive response of the heart to a variety of intrinsic and extrinsic stimuli, including hypertension, myocardial infarction, endocrine disorders, valvular diseases and so on. Although hypertrophy is initially beneficial, prolonged hypertrphy can ultimately become deleterious and result in cardiomyopathy, heart failure, and sudden death. Elucidation of the molecular mechanisms that activate and maintain the hypertrophic program represents a major challenge.Previous studies had demonstrated that FAK may function as a "scaffold" for the assembly of signaling complexes through the coordinated recruitment of other signaling and adaptor proteins. Activation of FAK (focal adhesion kinase) has been implicated in the progress of cardiomyocyte hypertrophy. FRNK (FAK-related nonkinase) is an alternate transcript of the C-terminal region of the FAK gene. Because FRNK does not possess catalytic activity but comprises the focal adhesion target (FAT) domain, FRNK can compete with FAK for focal adhesion binding site and act as an endogenous regulator of FAK activity. However, whether could FRNK inhibit cardiomyocytes hypertrophy via down-regulating key molecules involved in intracellular signal transduction? How about therapeutic effects of FRNK on pressure overload-induced cardiac hypertrophy? This study investigated the effect of FRNK on cardiomyocyte hypertrophy induced by angiotensin II and focused on the changes of relative signal molecules to reveal the possible anti-hypertrophy mechanisms of FRNK. Furthermore, we determined whether gene delivery of FRNK could inhibit a rat model of cardiac hypertrophy induced by abdominal aortic binding.MethodsFirstly, we designed a pair of unique primer according to the sequence of FRNK mRNA reported in NCBI. The upstream and downstream primers were added to BamH. I and EcoR I restriction sites, respectively. Using reverse transcriptase-polymerase chain reaction (RT-PCR) techniques, the full-length FRNK cDNA was obtained and then cloned to pcDNA3.1 vector by BamU I and EcoR I restriction sites. The correct plasmid of recombinant FRNK eukaryotic expression vector identified by the restriction analysis was further confirmed using an automated DNA sequencer and was subsequently compared with the published cDNA sequence.Secondly, Hypertrophy in neonatal rat cardiac myocytes was established with angiotensin II stimulation. And the pcDNA3.1-FRNK or pcDNA3.1 was respectively transfected into cardiomyocytes by lipofectamine? 2000. The surface area and mRNA expression of atrial natriuretic peptide (ANP) of myocytes were employed to detect cardiac hypertrophy. FRNK, phosphorylation levels of FAK (p-FAK), ERK1/2 (p-ERK1/2) and AKT (p-AKT), as well as total ERK1/2, AKT in variant treated cardiomyocytes were determined by Western blot.Finally, male Sprague-Dawley rats weighing 180 to 200 g were divided into 4 groups at random: 1) sham-operated group (n=9); 2) pressure-overloaded group (n=8); 3) FRNK group (n=10); 4) pcDNA3.1 group (n=9). Animals were performed the abdominal aortic banding operation. In the sham-operated group, the aorta was just isolated but not ligated. Rats in FRNK or pcDNA3.1 groups were monitored for 1 week after operations and then purified pcDNA3.1-FRNK or pcDNA3.1 were respectively injected into the tail vein at a dose of 4mg/kg body weight. 3 weeks later, these rats were received repeated injection in like manner. Pressure-overloaded group was subjected to aortic banding without injection. 7 weeks after aortic banding, cardiac function was assessed by hemodynamics. The ratios of heart weight to body weight (HW/BW) and left ventricle plus septum weight to body weight (LVW/BW) were calculated. Myocardial interstitial fibrosis was examined by picrosirius red staining. mRNA expression of ANP was evaluated by RT-PCR. Expressions of FAK phosphorylation and FRNK were determined by Western blot.ResultsWe successfully obtained FRNK cDNA using RT-PCR and it was confirmed by restrictive enzyme cutting and sequencing.In vitro, the surface area of myocytes and level of ANP mRNA were significantly increased under the stimulation of angiotensin II. But transient transfection with pcDNA3.1-FRNK in advance may reduce the surface area and expression of ANP mRNA of hypertrophic myocytes. The protein level of FRNK in FRNK group was obviously higher than that in other groups, but the p-FAK was dramatically decreased. The expressions of p-ERK1/2 and p-AKT in FRNK treated cardiomyocytes were dramatically decreased compared with that in angiotensin II induced cardiomyocytes, while different treatments had little effect on total ERK1/2 and AKT.In vivo, abdominal aortic banding significantly increased the rat HW/BW, LVW/BW, myocardial fibrosis, expression of ANP mRNA and decreased cardiac function compared with sham controls. Administration of pcDNA3.1-FRNK reduced the HW/BW, LVW/BW, and fibrosis, as well as ANP mRNA, and improved cardiac function compared with aortic-banded rats. Furthermore, FRNK level was higher while FAK Tyr³⁹⁷ phosphorylation was lower in the pcDNA3.1-FRNK treated rats compared with aortic-banded rats.ConclusionsThis study demonstrated thatâ‘  FRNK may inhibit angiotensin II -induced cardiomyocyte hypertrophy viadecreasing FAK activity and subsequently down-regulation the phosphorylationlevels of ERK1/2 and AKT. â‘¡ Eukaryotic expression vector pcDNA3.1-mediated FRNK gene delivery via vein significantly increased the expression of FRNK and blocked the phosphorylationlevel of FAK in hearts of rats with abdominal aortic banding.â‘¢ FRNK gene delivery could dramatically reduce cadiac fibrosis as well as improvecardiac function in pressure-overloaded cardiac hypertrophy rats.The present study suggested that FRNK may be a potential therapeutic target for the treatment of cardiac hypertrophy. FAK related non-kinase (FRNK) decelerates malignantproliferation of human breast carcinomacell line MDA-MB-435 in vitroBackground & ObjectiveBreast carcinoma is one of the most common cancers worldwide, especially in women. Identifying the breast carcinoma cell proliferation is essential to understand the mechanisms of human breast cancer progression and to develop more effective methods of diagnosis and management. Recently, many studies indicated that FAK was a new marker of human breast carcinoma. A continual FAK activation could enable carcinoma to proliferate. Previous experiments have approved that deviant regulation of cell cycle is one of the most important mechanisms of cell malignant proliferation. FAK is an important mediator of signal transduction pathways, and Ty³⁹⁷ in FAK plays a critical role in the cell cycle regulation. Inhibition of FAK397 phosphorylation may cause cell cycle arrest and subsequently suppress cell proliferation. FAK related non-kinase (FRNK) is an alternate transcript of the C-terminal region of the FAK gene. FRNK dose not possess catalytic activity but comprises the focal adhesion target (FAT) domain, so FRNK can compete with FAK for focal adhesion binding site and act as an endogenous regulator of FAK activity. But the negative contribution made by FRNK in the regulation of cell cycle progression is to date poorly understood. Base on the view of above-mentioned theoretical foundation, in the present study we used full-length FRNK cDNA successfully cloned in task 1^st as the research object, and examined the effect of FRNK on cell cycle progression. Furthermore, we sought to investigate the possible mechanism involved in inhibitory effect of FRNK on proliferation of human breast carcinoma cell line MDA-MB-435. MethodsLipofectamine^TM2000 was used to mediate transfection of pcDNA3.1-FRNK, pcDNA3.1-GFP and pcDNA3.1. Normal MDA-MB-435 cells were cultured as a negative control. The expression of FRNK and GFP were used to monitor transfection efficiency. The protein level of FAK phosphorylation at Tyr³⁹⁷ was also detected. 12 h, 24 h, 48 h or 72 h after the transfection, proliferative activities were evaluated by MTT method. And cell cycle was analysed by flow cytometry at 48 h after transfected with above plasmids. The expression of NF-κB p65 in nuclear extracts of the above transfected MDA-MB-435 cells were detected by Western blot.ResultsThe recombinant vector pcDNA3.1-FRNK may be transfected into MDA-MB-435 cells with high efficiency via Lipofectamine^TM2000. A maximal expression of FRNK in MDA-MB-435 cells was observed 48 h after transfected with pcDNA3.1-FRNK, while FAK phosphorylation at Tyr³⁹⁷ was dramatically decreased at the same time. The MTT results showed that the over-expression of FRNK gene inhibited proliferation of MDA-MB-435 cells. And this effect was time-dependent in a certain extent. Cell cycle analysis using flow cytometry displayed that transfection of FRNK gene significantly decreased the cell number of S/G2/M phase compared with the non-transfected cells (P <0.05). And the levels of NF-κB p65 protein in nuclear extracts of FRNK-transfected cells were decreased.ConclusionThe study suggested that over-expression of FRNK can decelerate the proliferation of MDA-MB-435 cells. And the phenomena may correlated with the competitive inhibition of FAK phosphorylation at Tyr³⁹⁷ and subsequently down-regulation of NF-κB p65 protein in nucleus, which could cause human breast carcinoma cell cycle arrest and finally suppress cell proliferation. This study provided substantial help for us to recognize and explore novel therapeutic strategies and develop new medicine for cancer.