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

Differences in Facial Expressions between Spontaneous and Posed Smiles: Automated Method by Action Units and Three-Dimensional Facial Landmarks

Research on emotion recognition from facial expressions has found evidence of different muscle movements between genuine and posed smiles. To further confirm discrete movement intensities of each facial segment, we explored differences in facial expressions between spontaneous and posed smiles with three-dimensional facial landmarks. Advanced machine analysis was adopted to measure changes in the dynamics of 68 segmented facial regions. A total of 57 normal adults (19 men, 38 women) who displayed adequate posed and spontaneous facial expressions for happiness were included in the analyses. The results indicate that spontaneous smiles have higher intensities for upper face than lower face. On the other hand, posed smiles showed higher intensities in the lower part of the face. Furthermore, the 3D facial landmark technique revealed that the left eyebrow displayed stronger intensity during spontaneous smiles than the right eyebrow. These findings suggest a potential application of landmark based emotion recognition that spontaneous smiles can be distinguished from posed smiles via measuring relative intensities between the upper and lower face with a focus on left-sided asymmetry in the upper region

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

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

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

Identification of 100 KD protein tyrosine-phosphorylation in response to myometrial stretch.

<p>A. Stretch-induced tyrosine phosphorylation in human myometrium. Term pregnant uterine smooth muscle strips unstretched or stretched to 2x slack length for 7 min. Coomassie blue staining of anti-phosphotyrosine immunoprecipitates separated by 10% SDS-polyacrylamide gel electrophoresis (n = 2). B. Mass spectrometric identification of the 100 kD band. The protein band was excised from the gel. There is a 281/911 (31%) sequence coverage with 23 peptides matched to alpha 4 actinin, (in yellow) and 19% sequence coverage with 13 peptides matched to alpha 1 actinin (not shown). C. Alpha actinin is tyrosine phosphorylated by stretch in human myometrium. Homogenates of stretched and unstretched term pregnant human uterine smooth muscle were immunoprecipitated (IP) with an anti-phospho-tyrosine antibody and western immunoblotted (IB) with an alpha-actinin antibody. The graph shows the average densitometry from 5 independent experiments. The inserts are typical western blots of immunoprecipitates. *p<0.05 compared to unstretched control samples.</p

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

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

<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