A role for focal adhesion kinase in facilitating the contractile responses of murine gastric fundus smooth muscles

Xie, Yeming; Han, Koon Hee; Grainger, Nathan; Li, Wen; Corrigan, Robert D.; Perrino, Brian A.

doi:10.1113/jp275406

Cited by 16 publications

(22 citation statements)

References 46 publications

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“…2015; Xie et al . 2018). Recently we demonstrated that membrane cholesterol and substrate stiffness coordinate to induce the remodelling of the cytoskeleton and alter the biomechanics of VSMCs (Sanyour et al .…”

Section: Introductionmentioning

confidence: 99%

Statin‐mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration

Sanyour

Rickel

et al. 2020

The Journal of Physiology

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Key points This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin‐mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin‐mediated cholesterol depletion. Proof is provided that statin‐mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin‐mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. Abstract Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG‐CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin‐mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin‐mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real‐time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time‐lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin‐mediated cholesterol depletion (‐27.8%) lowered VSMC migration distance on a fibronectin (FN)‐coated surface (‐14.8%) but not on a type 1 collagen (COL1)‐coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (‐46.6%) and ex vivo aortic ring contraction force (‐40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (‐24.5%) following statin‐mediated cholesterol depletion. Altogether, it is concluded that statin‐mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function.

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Section: Introductionmentioning

confidence: 99%

Statin‐mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration

Sanyour

Rickel

et al. 2020

The Journal of Physiology

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“…We previously found that PF-431396 inhibits the increase in CPI-17 T38 phosphorylation during EFS-evoked cholinergic contractions [21]. Because FAK is recruited to β1integrin in focal adhesions, we examined whether CPI-17 is also recruited and phosphorylated at focal adhesions.…”

Section: Effects Of Cholinergic Stimulation and Pf-431396 On β1integrmentioning

confidence: 96%

“…Gastric fundus smooth muscles were obtained by sharp dissection as described [21]. Each sheet of fundus smooth muscles was pinned out flat, cut into a rectangle of 10mm × 15mm, and then folded, in parallel with the circular muscles, into a rectangle of 10mm × 5mm.…”

Section: Contractile Responsesmentioning

confidence: 99%

“…We previously found that FAK is phosphorylated at the activation Y397, in response to EFSevoked cholinergic contractions of murine gastric fundus muscles [21]. Since FAK Y397 phosphorylation is associated with the recruitment of FAK to integrins within focal adhesions [54], we used isPLA to examine the localization of FAK and β1integrin in response to EFS-evoked cholinergic contractions.…”

Section: Effects Of Cholinergic Stimulation and The Fak Inhibitor Pf-mentioning

confidence: 99%

“…Tyrosine phosphorylation and activation of protein tyrosine kinase 2β (Pyk2) or focal adhesion kinase (FAK), along with the recruitment of other integrin-associated proteins to focal adhesions, occurs during contraction and force development [17,19,20]. We showed that mechanical force or tension is sufficient to activate FAK, and that FAK is involved in the activation of the PKC-CPI-17 Ca 2+ sensitization pathway in gastric fundus smooth muscles, suggesting that myofilament Ca 2+ sensitization mechanisms play a role in myofilament function at focal adhesions [21]. These actin filament modifications and remodeling are thought to facilitate the polymerization of cortical cytoskeletal actin filaments to increase the stability of focal adhesions in the membrane, allowing for the force generated by actin filaments attached to the cytoplasmic domains of β1integrin by talin to be transmitted to the connective tissue of the extracellular matrix [22][23][24][25].…”

Section: Introductionmentioning

confidence: 97%

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Quantitative in situ proximity ligation assays examining protein interactions and phosphorylation during smooth muscle contractions

Xie

Perrino

2019

Analytical Biochemistry

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Antibody-based in situ proximity ligation assays (isPLA) have the potential to study protein phosphorylation and protein interactions with spatial resolution in intact tissues. However, the application of isPLA at the tissue level is limited by a lack of appropriate positive and negative controls and the difficulty in accounting for changes in tissue shape. Here we demonstrate a set of experimental and computational approaches using gastric fundus smooth muscles to improve the validity of quantitative isPLA. Appropriate positive and negative biological controls and PLA technical controls were selected to ensure experimental rigor. To account for changes in morphology between relaxed and contracted smooth muscles, target PLA spots were normalized to smooth muscle myosin light chain 20 PLA spots or the cellular cross-sectional areas. We describe the computational steps necessary to filter out false-positive improperly sized spots and set the thresholds for counting true positive PLA spots to quantify the PLA signals. We tested our approach by examining protein phosphorylation and protein interactions in smooth muscle myofilament Ca 2+ sensitization pathways from resting and contracted gastric fundus smooth muscles. In conclusion, our tissue-level isPLA method enables unbiased quantitation of protein phosphorylation and protein-protein interactions in intact smooth muscle tissues, suggesting the potential for quantitative isPLA applications in other types of intact tissues.

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Mfge8 attenuates human gastric antrum smooth muscle contractions

Olseen

Xie

et al. 2021

J Muscle Res Cell Motil

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Coordinated gastric smooth muscle contraction is critical for proper digestion and is adversely affected by a number of gastric motility disorders. In this study we report that the secreted protein Mfge8 (milk fat globule-EGF factor 8) inhibits the contractile responses of human gastric antrum muscles to cholinergic stimuli by reducing the inhibitory phosphorylation of the MYPT1 (myosin phosphatase-targeting subunit (1) subunit of MLCP (myosin light chain phosphatase), resulting in reduced LC20 (smooth muscle myosin regulatory light chain (2) phosphorylation. Mfge8 reduced the agonist-induced increase in the F-actin/G-actin ratios of β-actin and γ-actin1. We show that endogenous Mfge8 is bound to its receptor, α8β1 integrin, in human gastric antrum muscles, suggesting that human gastric antrum muscle mechanical responses are regulated by Mfge8. The regulation of gastric antrum smooth muscles by Mfge8 and α8 integrin functions as a brake on gastric antrum mechanical activities. Further studies of the role of Mfge8 and α8 integrin in regulating gastric antrum function will likely reveal additional novel aspects of gastric smooth muscle motility mechanisms.

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A role for focal adhesion kinase in facilitating the contractile responses of murine gastric fundus smooth muscles

Cited by 16 publications

References 46 publications

Statin‐mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration

Statin‐mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration

Quantitative in situ proximity ligation assays examining protein interactions and phosphorylation during smooth muscle contractions

Mfge8 attenuates human gastric antrum smooth muscle contractions

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