ERK3 is involved in regulating cardiac fibroblast function.

ERK3/MAPK6 activates MAP kinase-activated protein kinase (MK)-5 in selected cell types. Male MK5 haplodeficient mice show reduced hypertrophy and attenuated increase in Col1a1 mRNA in response to increased cardiac afterload. In addition, MK5 deficiency impairs cardiac fibroblast function. This study determined the effect of reduced ERK3 on cardiac hypertrophy following transverse aortic constriction (TAC) and fibroblast biology in male mice. Three weeks post-surgery, ERK3, but not ERK4 or p38α, co-immunoprecipitated with MK5 from both sham and TAC heart lysates. The increase in left ventricular mass and myocyte diameter was lower in TAC-ERK3+/- than TAC-ERK3+/+ hearts, whereas ERK3 haploinsufficiency did not alter systolic or diastolic function. Furthermore, the TAC-induced increase in Col1a1 mRNA abundance was diminished in ERK3+/- hearts. ERK3 immunoreactivity was detected in atrial and ventricular fibroblasts but not myocytes. In both quiescent fibroblasts and "activated" myofibroblasts isolated from adult mouse heart, siRNA-mediated knockdown of ERK3 reduced the TGF-ß-induced increase in Col1a1 mRNA. In addition, intracellular type 1 collagen immunoreactivity was reduced following ERK3 depletion in quiescent fibroblasts but not myofibroblasts. Finally, knocking down ERK3 impaired motility in both atrial and ventricular myofibroblasts. These results suggest that ERK3 plays an important role in multiple aspects of cardiac fibroblast biology.

Protein tyrosine phosphatase 1B regulates miR-208b-argonaute 2 association and thyroid hormone responsiveness in cardiac hypertrophy.

Increased production of reactive oxygen species plays an essential role in the pathogenesis of several diseases, including cardiac hypertrophy. In our search to identify redox-sensitive targets that contribute to redox signaling, we found that protein tyrosine phosphatase 1B (PTP1B) was reversibly oxidized and inactivated in hearts undergoing hypertrophy. Cardiomyocyte-specific deletion of PTP1B in mice (PTP1B cKO mice) caused a hypertrophic phenotype that was exacerbated by pressure overload. Furthermore, we showed that argonaute 2 (AGO2), a key component of the RNA-induced silencing complex, was a substrate of PTP1B in cardiomyocytes and in the heart. Our results revealed that phosphorylation at Tyr393 and inactivation of AGO2 in PTP1B cKO mice prevented miR-208b-mediated repression of thyroid hormone receptor-associated protein 1 (THRAP1; also known as MED13) and contributed to thyroid hormone-mediated cardiac hypertrophy. In support of this conclusion, inhibiting the synthesis of triiodothyronine (T3) with propylthiouracil rescued pressure overload-induced hypertrophy and improved myocardial contractility and systolic function in PTP1B cKO mice. Together, our data illustrate that PTP1B activity is cardioprotective and that redox signaling is linked to thyroid hormone responsiveness and microRNA-mediated gene silencing in pathological hypertrophy.

MK2-Deficient Mice Are Bradycardic and Display Delayed Hypertrophic Remodeling in Response to a Chronic Increase in Afterload.

Background Mitogen-activated protein kinase-activated protein kinase-2 (MK2) is a protein serine/threonine kinase activated by p38α/ß. Herein, we examine the cardiac phenotype of pan MK2-null (MK2-/-) mice. Methods and Results Survival curves for male MK2+/+ and MK2-/- mice did not differ (Mantel-Cox test, P=0.580). At 12 weeks of age, MK2-/- mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, aging was not associated with an abnormal reduction in diastolic function. Both R-R interval and P-R segment durations were prolonged in MK2-deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2+/+ and MK2-/- mice. MK2-/- mice had lower body temperature and an age-dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a, Acadm, Lipe, and Ucp3, were increased in hearts from MK2-/- mice. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+ sensitivity to mitochondrial permeability transition pore opening. Eight weeks of pressure overload increased left ventricular mass in MK2+/+ and MK2-/- mice; however, after 2 weeks the increase was significant in MK2+/+ but not MK2-/- mice. Finally, the pressure overload-induced decrease in systolic function was attenuated in MK2-/- mice 2 weeks, but not 8 weeks, after constriction of the transverse aorta. Conclusions Collectively, these results implicate MK2 in (1) autonomic regulation of heart rate, (2) cardiac mitochondrial function, and (3) the early stages of myocardial remodeling in response to chronic pressure overload.

A putative protein-RNA complex regulates posttranscriptional processing of cytochrome P450 aromatase (CYP19A1) in bovine granulosa cells.

Follicle growth and granulosa cell health are dependent on the secretion of estradiol from granulosa cells. Estradiol is synthesized from androgen precursor by cytochrome P450 aromatase (CYP19A1), and in cattle CYP19A1 messenger RNA has a short half-life but a long (3.5 kb) 3'-untranslated region (3'UTR), suggesting that posttranscriptional regulation may be important for control of enzyme activity. We tested this hypothesis by inserting the CYP19A1 3'UTR and fragments thereof into a reporter vector between the end of the luciferase coding region and the polyadenylation signal. The full-length aromatase 3'UTR suppressed luciferase activity to 10% of control levels, and smaller fragments showed that this inhibitory activity lies between +926 and +1134 of the 3'UTR. Protein-RNA cross-linking experiments revealed that these 3'UTR fragments formed an RNA-protein complex of approximately 70 kDa that was present in granulosa cells but not in corpus luteum, lung, liver, kidney, pancreas, or bladder extracts. The RNA-binding activity was specific to the 3'UTR, as shown by competition experiments with unlabeled RNA, and was present only in 3'UTR constructs that inhibited luciferase activity. These data suggest that posttranscriptional regulation is an important component of the control of CYP19A1 expression and involves protein binding to a specific sequence in the 3'UTR.

Transcript levels for extracellular matrix proteins are altered in MK5-deficient cardiac ventricular fibroblasts.

MK5 is a protein serine/threonine kinase activated by p38 MAPK and the atypical MAPKs ERK3 and ERK4. Although little is known of the physiological role of MK5 in the heart, both hypertrophic growth and the increase in collagen 1-α1 mRNA induced by increased afterload are attenuated in hearts of MK5 haploinsufficient (MK5+/-) mice. MK5 transcripts are detected at high levels in the left ventricular myocardium; however, MK5 immunoreactivity is detected in adult cardiac fibroblasts, but not myocytes. The present study was to determine if MK5 has a potential role in remodeling of the extracellular matrix. Ventricular fibroblasts were isolated from MK5+/+, MK5+/-, or MK5-/- mice and maintained in culture on either compliant (8 kPa) or rigid substrates to obtain quiescent fibroblasts or activated myofibroblasts, respectively. In quiescent fibroblasts, reduced MK5 had little effect: BMP7 and TGF-ß1 mRNA was increased in MK5+/- and MK5-/-.cells, respectively. Ang-II altered the abundance of numerous transcripts in an MK5-sensitive manner. Both collagen 1-α1 mRNA and secreted type 1 collagen immunoreactivity were increased by Ang-II in wild type but not MK5-deficient fibroblasts. The effects of deleting MK5 were quite different in myofibroblasts: both the abundance of collagen 1-α1 mRNA and secreted type 1 collagen immunoreactivity elevated in the absence of added Ang-II and addition of Ang-II failed to evoke a further increase in either. In addition, whereas type I collagen immunoreactivity was distributed throughout the cytosol of wild-type myofibroblasts, it was perinuclear in MK5-/- myofibroblasts. Furthermore, in MK5-deficient myofibroblasts the abundance of collagen 3-α2, Timp3, Smad 6, Smad 7, TGF-ß3, and snail homolog 1 transcripts was increased whereas integrin ß3, latent TGF-ß binding protein 1, thrombospondin 1, hepatocyte growth factor, and interleukin 13 were decreased. Finally, fibroblast contraction was decreased upon knocking down MK5. These results indicate that MK5 may be involved in fibroblast-mediated regulation of extracellular matrix homeostasis.

MK5 haplodeficiency decreases collagen deposition and scar size during post-myocardial infarction wound repair.

MK5 is a protein serine/threonine kinase activated by p38, ERK3, and ERK4 MAPKs. MK5 mRNA and immunoreactivity are detected in mouse cardiac fibroblasts, and MK5 haplodeficiency attenuates the increase in collagen 1-α1 mRNA evoked by pressure overload. The present study examined the effect of MK5 haplodeficiency on reparative fibrosis following myocardial infarction (MI). Twelve-week-old MK5+/- and wild-type littermate (MK5+/+) mice underwent ligation of the left anterior descending coronary artery (LADL). Surviving mice were euthanized 8 or 21 days post-MI. Survival rates did not differ significantly between MK5+/+ and MK5+/- mice, with rupture of the LV wall being the primary cause of death. Echocardiographic imaging revealed similar increases in LV end-diastolic diameter, myocardial performance index, and wall motion score index in LADL-MK5+/+ and LADL-MK5+/- mice. Area at risk did not differ between LADL-MK5+/+ and LADL-MK5+/- hearts. In contrast, infarct size, scar area, and scar collagen content were reduced in LADL-MK5+/- hearts. Immunohistochemical analysis of mice experiencing heart rupture revealed increased MMP-9 immunoreactivity in the infarct border zone of LADL-MK5+/- hearts compared with LADL-MK5+/+. Although inflammatory cell infiltration was similar in LADL-MK5+/+ and LADL-MK5+/- hearts, angiogenesis was more pronounced in the infarct border zone of LADL-MK5+/- mice. Characterization of ventricular fibroblasts revealed reduced motility and proliferation in fibroblasts isolated from MK5-/- mice compared with those from both wild-type and haplodeficient mice. siRNA-mediated knockdown of MK5 in fibroblasts from wild-type mice also impaired motility. Hence, reduced MK5 expression alters fibroblast function and scar morphology but not mortality post-MI. NEW & NOTEWORTHY MK5/PRAK is a protein serine/threonine kinase activated by p38 MAPK and/or atypical MAPKs ERK3/4. MK5 haplodeficiency reduced infarct size, scar area, and scar collagen content post-myocardial infarction. Motility and proliferation were reduced in cultured MK5-null cardiac myofibroblasts.

MK5 haplodeficiency attenuates hypertrophy and preserves diastolic function during remodeling induced by chronic pressure overload in the mouse heart.

MAPK-activated protein kinase-5 (MK5) is a protein serine/threonine kinase that is activated by p38 MAPK and the atypical MAPKs ERK3 and ERK4. The physiological function(s) of MK5 remains unknown. Here, we examined the effect of MK5 haplodeficiency on cardiac function and myocardial remodeling. At 12 wk of age, MK5 haplodeficient mice (MK5+/-) were smaller than age-matched wild-type littermates (MK5+/+), with similar diastolic function but reduced systolic function. Transverse aortic constriction (TAC) was used to induce chronic pressure overload in 12-wk-old male MK5+/- and MK5+/+ mice. Two weeks post-TAC, heart weight-to-tibia length ratios were similarly increased in MK5+/- and MK5+/+ hearts, as was the abundance of B-type natriuretic peptide and ß-myosin heavy chain mRNA. Left ventricular ejection fraction was reduced in both MK5+/+ and MK5+/- mice, whereas regional peak systolic tissue velocities were reduced and isovolumetric relaxation time was prolonged in MK5+/+ hearts but not in MK5+/- hearts. The TAC-induced increase in collagen type 1-α1 mRNA observed in MK5+/+ hearts was markedly attenuated in MK5+/- hearts. Eight weeks post-TAC, systolic function was equally impaired in MK5+/+ and MK5+/- mice. In contrast, the increase in E wave deceleration rate and progression of hypertrophy observed in TAC MK5+/+ mice were attenuated in TAC MK5+/- mice. MK5 immunoreactivity was detected in adult fibroblasts but not in myocytes. MK5+/+, MK5+/-, and MK5-/- fibroblasts all expressed α-smooth muscle actin in culture. Hence, reduced MK5 expression in cardiac fibroblasts was associated with the attenuation of both hypertrophy and development of a restrictive filling pattern during myocardial remodeling in response to chronic pressure overload.NEW & NOTEWORTHY MAPK-activated protein kinase-5 (MK5)/p38-regulated/activated protein kinase is a protein serine/threonine kinase activated by p38 MAPK and/or the atypical MAPKs ERK3 and ERK4. MK5 immunoreactivity was detected in adult ventricular fibroblasts but not in myocytes. MK5 haplodeficiency attenuated the progression of hypertrophy, reduced collagen type 1 mRNA, and protected diastolic function in response to chronic pressure overload.

Nitric oxide synthase activity is critical for the preovulatory epidermal growth factor-like cascade induced by luteinizing hormone in bovine granulosa cells.

In rabbits and rodents, nitric oxide (NO) is generally considered to be critical for ovulation. In monovulatory species, however, the importance of NO has not been determined, nor is it clear where in the preovulatory cascade NO may act. The objectives of this study were (1) to determine if nitric oxide synthase (NOS) enzymes are regulated by luteinizing hormone (LH) and (2) to determine if and where endogenous NO is critical for expression of genes essential for the ovulatory cascade in bovine granulosa cells in serum-free culture. Time- and dose-response experiments demonstrated that LH had a significant stimulatory effect on endothelial NOS (NOS3) mRNA abundance, but in a prostaglandin-dependent manner. NO production was stimulated by LH before a detectable increase in NOS3 mRNA levels was observed. Pretreatment of cells with the NOS inhibitor L-NAME blocked the effect of LH on the epidermal growth factor (EGF)-like ligands epiregulin and amphiregulin, as well as prostaglandin-endoperoxide synthase-2 mRNA abundance and protein levels. Similarly, EGF treatment increased mRNA encoding epiregulin, amphiregulin, and the early response gene EGR1, and this was inhibited by pretreatment with L-NAME. Interestingly, pretreatment with L-NAME had no effect on either ERK1/2 or AKT activation. Taken together, these results suggest that endogenous NOS activity is critical for the LH-induced ovulatory cascade in granulosa cells of a monotocous species and acts downstream of EGF receptor activation but upstream of the EGF-like ligands.

Factors regulating the bovine, caprine, rat and human ovarian aromatase promoters in a bovine granulosa cell model.

The ovarian promoter of the primate and rodent genes encoding cytochrome P450 aromatase (CYP19A1) are robustly responsive to forskolin in luteinized cell models, whereas the ruminant ovarian promoter is minimally active. We explored this discrepancy by investigating the activity of the bovine ovarian promoter in two bovine granulosa cell models, luteinizing and non-luteinizing cells in vitro. In non-luteinizing cells, both FSH and IGF1 increased abundance of transcripts derived from the ovarian promoter. Comparison of the activity of promoters of several species in response to transcription factors (forskolin, NR5A2, FOXL2) in luteinizing cells demonstrated that a rat ovarian promoter-luciferase reporter was regulated mainly by forskolin (18-fold increase over basal expression) and addition of NR5A2 or FOXL2 had no further effect. Activity of a human promoter was significantly increased by NR5A2 plus forskolin (153-fold) compared with forskolin alone (71-fold over basal); addition of FOXL2 did not significantly increase promoter activity. Forskolin alone provoked minor activation of caprine and bovine promoter reporters (3-fold over basal), and addition of NR5A2 increased activity (7- to 11-fold). When forskolin, NR5A2 and FOXL2 treatments were combined, the activity of the caprine and bovine promoters increased to 20- and 34-fold, respectively. These data suggest that a major reason why CYP19A1 is not expressed in luteinized cells (and the corpus luteum) of ruminants may be the stimulatory effect of FOXL2, which does not appear to be the case in the human and rat.