The actin-associated protein palladin is required for development of normal contractile properties of smooth muscle cells derived from embryoid bodies

Li Jin, Tadashi Yoshida, Ruoya Ho, Gary K. Owens, Avril V. Somlyo

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Palladin is a widely expressed actin-associated protein localized at stress fibers, focal adhesions, and other actin-based structures, playing a significant role in cell adhesion and cell motility. Knockout of Palladin in mice is embryonic lethal, demonstrating the importance of Palladin in development yet its role in the vasculature is not known. In the present study, smooth muscle cell (SMC) markers, such as myosin, actin, caldesmon, calponin, and LPP, were down-regulated in embryoid bodies (EBs) derived from embryonic stem cells lacking Palladin. Transgenic embryonic stem cell lines were generated that stably expressed a puromycin-resistance gene under the control of a SM α-actin (SMA) promoter. Negative selection was then used to purify SMCs from EBs. Purified SMCs expressing multiple SMC markers were designated APSCs (SMA-puromycin-selected cells). Palladin null APSCs express significantly less myosin, actin, calponin, and h-caldesmon. The filamentous (F) to globular (G) actin ratio, known to regulate myocardin family transcription factors, was also decreased. Palladin null APSCs showed increased cell adhesion and decreased cell motility. Importantly, Palladin null APSCs within collagen gels generated less maximum contractile force when stimulated with endothelin-1, sphingosine 1-phosphate (S1P), and thrombin. Myosin light chains (MLC20) were phosphorylated by lysophosphatidic acid to the same extent in Palladin null and wild type APSCs but myosin content/total protein was reduced by >50%, consistent with the observed decreases in contractility. All together, these results suggest that Palladin is essential for expression of the full complement of contractile proteins necessary for optimal force development of SMCs derived from EBs.

Original languageEnglish
Pages (from-to)2121-2130
Number of pages10
JournalJournal of Biological Chemistry
Volume284
Issue number4
DOIs
Publication statusPublished - 2009 Jan 23
Externally publishedYes

Fingerprint

Embryoid Bodies
Smooth Muscle Myocytes
Muscle
Actins
Cells
Myosins
Proteins
Calmodulin-Binding Proteins
Puromycin
Cell adhesion
Embryonic Stem Cells
Stem cells
Cell Adhesion
Cell Movement
Contractile Proteins
Stress Fibers
Myosin Light Chains
Focal Adhesions
Endothelin-1
Thrombin

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

The actin-associated protein palladin is required for development of normal contractile properties of smooth muscle cells derived from embryoid bodies. / Jin, Li; Yoshida, Tadashi; Ho, Ruoya; Owens, Gary K.; Somlyo, Avril V.

In: Journal of Biological Chemistry, Vol. 284, No. 4, 23.01.2009, p. 2121-2130.

Research output: Contribution to journalArticle

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abstract = "Palladin is a widely expressed actin-associated protein localized at stress fibers, focal adhesions, and other actin-based structures, playing a significant role in cell adhesion and cell motility. Knockout of Palladin in mice is embryonic lethal, demonstrating the importance of Palladin in development yet its role in the vasculature is not known. In the present study, smooth muscle cell (SMC) markers, such as myosin, actin, caldesmon, calponin, and LPP, were down-regulated in embryoid bodies (EBs) derived from embryonic stem cells lacking Palladin. Transgenic embryonic stem cell lines were generated that stably expressed a puromycin-resistance gene under the control of a SM α-actin (SMA) promoter. Negative selection was then used to purify SMCs from EBs. Purified SMCs expressing multiple SMC markers were designated APSCs (SMA-puromycin-selected cells). Palladin null APSCs express significantly less myosin, actin, calponin, and h-caldesmon. The filamentous (F) to globular (G) actin ratio, known to regulate myocardin family transcription factors, was also decreased. Palladin null APSCs showed increased cell adhesion and decreased cell motility. Importantly, Palladin null APSCs within collagen gels generated less maximum contractile force when stimulated with endothelin-1, sphingosine 1-phosphate (S1P), and thrombin. Myosin light chains (MLC20) were phosphorylated by lysophosphatidic acid to the same extent in Palladin null and wild type APSCs but myosin content/total protein was reduced by >50{\%}, consistent with the observed decreases in contractility. All together, these results suggest that Palladin is essential for expression of the full complement of contractile proteins necessary for optimal force development of SMCs derived from EBs.",
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