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PKC regulation of microfilament network organization in keratinocytes defined by a pharmacological study with PKC
B Masson-Gadais1, P Salers, P Bongrand
1Laboratoire d'Immunologie, Hôpital Sainte Marguerite, Unité INSERM U387, Marseille, France.
Abstract:
The modulation by PKC activators and inhibitors of adhesion, spreading, migration, actin cytoskeleton organization, and focal complex formation in keratinocytes attaching to type I collagen was studied. Two actin microfilament networks, stress fibers and cortical actin, could be distinguished on the basis of cellular distribution and opposite regulation by growth factors, tyrosine kinase inhibitors, and PKC activators. Stress fiber formation was stimulated by growth factors and by PMA (100 ng/ml) and these stimulations were blocked by tyrosine kinase inhibitors (0.3 mM genistein and 1 microM herbimycin A). By contrast, the cortical network occurred in quiescent cells, was unaffected by tyrosine kinase inhibitors, and was broken down after PKC activation by PMA. Spreading, migration, and actin polymerization were completely blocked while adhesion efficacy was significantly decreased by three specific PKC inhibitors. Half-inhibition of migration was obtained with 0.025, 1, and 3 microM concentrations of calphostin C, chelerytrine chloride, and D-erythrosphingosine, respectively, which are concentrations close to those known to inhibit the PKC kinase function in vitro. Paxillin clustering, which was observed even in the presence of tyrosine kinase inhibitors, disappeared only when actin polymerization was completely impaired, i.e., in cells treated with PKC inhibitors or with both tyrosine kinase inhibitors and PMA, which indicated that focal complex formation was highly dependent on microfilament reorganization. The analysis of these data underscores a major regulation function of PKC in the molecular events involved in growth factor and adhesion-dependent regulation of microfilament dynamics.
Insights
Protein Kinase C (PKC) regulates keratinocyte adhesion, migration, and actin cytoskeleton organization. PKC activation disrupts cortical actin, while inhibition blocks cell spreading and migration, highlighting its key role in microfilament dynamics.
Area of Science:
- Cell Biology
- Biochemistry
- Dermatology
Background:
- Keratinocyte adhesion and migration are crucial for skin integrity and wound healing.
- Actin cytoskeleton dynamics play a vital role in cell morphology, movement, and focal adhesion formation.
- Protein Kinase C (PKC) is implicated in various cellular signaling pathways, including those affecting the cytoskeleton.
Purpose of the Study:
- To investigate the role of Protein Kinase C (PKC) in modulating keratinocyte adhesion, spreading, migration, and actin cytoskeleton organization.
- To differentiate the functions of two distinct actin microfilament networks (stress fibers and cortical actin) in response to PKC activators and inhibitors.
Main Methods:
- Utilized PKC activators (PMA) and specific inhibitors (calphostin C, chelerytrine chloride, D-erythrosphingosine) to treat keratinocytes.
- Examined effects on cell adhesion, spreading, migration, actin polymerization, and focal complex formation.
- Distinguished between stress fiber and cortical actin networks based on their distribution and regulation.
Main Results:
- PKC activation by PMA stimulated stress fiber formation but disrupted cortical actin.
- PKC inhibitors significantly decreased adhesion efficacy and completely blocked spreading and migration.
- Focal complex formation, indicated by paxillin clustering, was dependent on microfilament reorganization and impaired by PKC inhibition.
Conclusions:
- PKC plays a major regulatory role in the molecular events governing microfilament dynamics.
- PKC signaling is critical for growth factor and adhesion-dependent regulation of keratinocyte behavior.
- Targeting PKC may offer therapeutic strategies for conditions involving impaired keratinocyte migration and wound healing.