Stretch Activates Human Myometrium via ERK, Caldesmon and Focal Adhesion Signaling

An incomplete understanding of the molecular mechanisms responsible for myometrial activation from the quiescent pregnant state to the active contractile state during labor has hindered the development of effective therapies for preterm labor. Myometrial stretch has been implicated clinically in the initiation of labor and the etiology of preterm labor, but the molecular mechanisms involved in the human have not been determined. We investigated the mechanisms by which gestation-dependent stretch contributes to myometrial activation, by using human uterine samples from gynecologic hysterectomies and Cesarean sections. Here we demonstrate that the Ca requirement for activation of the contractile filaments in human myometrium increases with caldesmon protein content during gestation and that an increase in caldesmon phosphorylation can reverse this inhibitory effect during labor. By using phosphotyrosine screening and mass spectrometry of stretched human myometrial samples, we identify 3 stretch-activated focal adhesion proteins, FAK, p130Cas, and alpha actinin. FAK-Y397, which signals integrin engagement, is constitutively phosphorylated in term human myometrium whereas FAK-Y925, which signals downstream ERK activation, is phosphorylated during stretch. We have recently identified smooth muscle Archvillin (SmAV) as an ERK regulator. A newly produced SmAV-specific antibody demonstrates gestation-specific increases in SmAV protein levels and stretch-specific increases in SmAV association with focal adhesion proteins. Thus, whereas increases in caldesmon levels suppress human myometrium contractility during pregnancy, stretch-dependent focal adhesion signaling, facilitated by the ERK activator SmAV, can contribute to myometrial activation. These results suggest that focal adhesion proteins may present new targets for drug discovery programs aimed at regulation of uterine contractility

Phosphorylation-dependent BRD4 dimerization and implications for therapeutic inhibition of BET family proteins.

Funder: AstraZenecaFunder: AstraZeneca postdoc fundBromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains. Phosphorylation by casein kinase II (CK2) regulates BRD4 function, is necessary for active transcription and is involved in resistance to BRD4 drug inhibition in triple-negative breast cancer. Here, we provide the first biophysical analysis of BRD4 phospho-regulation. Using integrative structural biology, we show that phosphorylation by CK2 modulates the dimerization of human BRD4. We identify two conserved regions, a coiled-coil motif and the Basic-residue enriched Interaction Domain (BID), essential for the BRD4 structural rearrangement, which we term the phosphorylation-dependent dimerization domain (PDD). Finally, we demonstrate that bivalent inhibitors induce a conformational change within BRD4 dimers in vitro and in cancer cells. Our results enable the proposal of a model for BRD4 activation critical for the characterization of its protein-protein interaction network and for the development of more specific therapeutics

SmAV protein content increases with gestation in both the human and the rat.

<p>A. Specificity of the anti SmAV antibody. B. Gestation-dependent increase in SmAV expression in human myometrium. Densitometry analysis of smooth muscle Archvillin (SmAV) protein levels from immunoblots of human myometrium from nonpregnant (NP) and pregnant women. A typical blot is shown on the top. *p<0.05 vs. NP. n = 5–7 samples in each group. C. Gestation-dependent increase in SmAV expression in rat myometrium. A typical blot is shown on the top. *p<0.05 compared to NP, **p<0.01 compared to NP. ++ p<0.01 compared to D16. n = 4 in each group. NP, nonpregnant, D16 and D20, pregnant day 16 or day 20.</p

Smooth muscle Archvillin (SmAV) in pregnant human myometrium is localized with vinculin at cell surface.

<p>Term, not-in-labor, human uterine smooth muscle strips were microdissected and stretched to 2 fold slack length for 7 minutes. The fresh frozen sections (10 sections for each sample) were stained with rabbit anti-SmAV and mouse anti-vinculin. Alexa-dye-conjugated goat secondary antibodies were used. Left column, SmAV (red); middle column vinculin (green); right column, merged image. Top panels, scale bar, 20 µm. Lower 3 panels, expanded magnification of boxed area in top merged image. SmAV localizes with the dense plaque maker vinculin (inset, white arrows) at cell surface. Scale bar, 10 µm, n = 3.</p

Stretch of human myometrium directly increases <i>h-</i>CaD and ERK phosphorylation.

<p>A. h-CaD phosphorylation increases in response to stretch. Phosphorylated p-<i>h</i>-CaD is normalized to <i>h-</i>CaD protein level. *p<0.05 and ** p = 0.01 compared to resting sample (by ANOVA). n = 4 samples in each group. B. Stretch increases ERK2 phosphorylation. p-ERK2 signals are normalized to the total ERK2 protein levels. *p<0.05 compared to resting samples (by ANOVA). n = 4–5 samples in each group.</p

<i>In vitro</i> stretch increases contractility in pregnant myometrium.

<p>Uterine contractility was measured and quantified as the area under curve (AUC). AUC is the integral of the <i>active</i> force over a period time of 2 or 7 minutes before and after stretch. Dotted line indicates level of passive stretch. Data presented in the bar graph were collected from 8 term, not in labor, pregnant human myometrial samples and total 17 smooth muscle strips. ++ p<0.01 compared to the AUC of spontaneous contraction before 2 minute-stretch. ** p<0.01 compared to the AUC of spontaneous contraction before 7 minute-stretch. An insert was the representative myograph recording of contractile force from term pregnant human myometrium in response to <i>in vitro</i> stretch.</p

Gestation-dependent changes in CaD content, calcium sensitivity and CaD phosphorylation.

<p>A. <i>h-</i>CaD expression increases during pregnancy. n = 3–7 samples in each group. * p<0.05 compared to NP. NP: nonpregnant. A single representative blot is illustrated on the top. To minimize the variation of the signal intensity between different Western blotting experiments, a reference sample was used in each blot for normalization since multiple samples were used from different pregnant women. B. Myometrial calcium sensitivity decreases with gestation. Human nonpregnant and pregnant not in labor uterine strips were permeabilized with alpha-toxin. Contractile force at 10⁻⁷ M calcium is expressed as a percentage of contraction induced by 10⁻⁵ M calcium. n = 3–5 samples in each group ** p<0.001 compared to NP. C. Increased CaD phosphorylation at Ser⁷⁸⁹ occurs only with the onset of labor. Phosphorylated p-<i>h</i>-CaD is normalized to <i>h-</i>CaD protein level. A single representative blot is included on the top. NP: nonpregnant, NIL: term, not in labor, IL: term, in labor. The samples used in experiments for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007489#pone-0007489-g001" target="_blank">Fig 1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007489#pone-0007489-g001" target="_blank">Fig 1C</a> are different. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007489#pone-0007489-g001" target="_blank">Figure 1C</a> is an independent experiment. *p<0.05 vs. NP. n = 5–7 samples in each group.</p

Identification of p125 as FAK and p130 as Cas.

<p>A. Stretch selectively activates FAK at tyrosine site 925, not site 397. Term pregnant uterine smooth muscle strips were unstretched or stretched to 2x slack length at indicated time period. The tissue homogenates were probed with FAK site-specific antibodies. Phospho-FAK signals are normalized to the total FAK protein or alpha-Tubulin. *p<0.05 compared to unstretched control samples (by ANOVA). n = 3–5 in each group. Typical blots were shown on the top. Both FAK total protein antibody and FAK-Y925 phospho antibody are rabbit polyclonal antibodies, thus, a-tubulin mouse monoclonal antibody was chosen for normalization of the FAK-Y925 signal. B. Stretch activates pCas130. Phospho-Cas signals are normalized to Cas protein levels and expressed as p-Cas/Cas ratios. *p<0.05 compared to unstretched control samples (by ANOVA). n = 3–5 samples in each group.</p

SmAV co-immunoprecipitates with phosphotyrosine, ERK and FAK.

<p>A. Myometrial SmAV associates with tyrosine-phosphorylated proteins in a stretch-dependent manner. Stretch and unstretched term pregnant human uterine smooth muscle homogenates were immunoprecipitated (IP) with anti-phospho-tyrosine antibody and immunoblotted (IB) with SmAV antibody. **p<0.01 compared to unstretched control samples. The insert is a typical SmAV western blot with IP samples. n = 5. B. Myometrial SmAV associates with ERK protein in a stretch- dependent manner. Stretched and unstretched term samples were immunoprecipitated with anti- ERK antibody and immunoblotted with anti-SmAV antibody. SmAV densitometry was normalized to input signals. n = 6. **p<0.01, ***p<0.001. C. Confirmation of stretch induced ERK phosphorylation in human myometrium. Stretch and unstretched samples were immunoprecipitated with anti- ERK antibody and immunoblotted with phospho-ERK antibody. The p-ERK2 infrared signals were also normalized to input signals. n = 6. **p<0.01. D. Myometrial ERK associates with FAK in a stretch-dependent manner. Stretch and unstretched samples homogenates were immunoprecipitated with anti- ERK antibody and immunoblotted with phospho-FAK⁹²⁵ antibody. The p-FAK infrared signals were normalized to input signals. n = 6. ***p<0.001</p

Stretch-induced tyrosine phosphorylation in human myometrium.

<p>A. A typical anti-phosphotyrosine immunoblot. Term pregnant uterine smooth muscle strips unstretched or stretched to 2x slack length for 2 min. The tissue homogenates were immunoblotted with an anti-tyrosine phosphorylation antibody. B–D. Term pregnant uterine smooth muscle strips were unstretched (rest) or stretched to 2x slack length at indicated time period. Average densitometry of phospho-tyrosine bands of 130 kD, 125 kD and 100 kD. n = 3–5 samples in each group.</p