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. 2010 Jan;298(1):C46-55.
doi: 10.1152/ajpcell.00387.2009. Epub 2009 Nov 4.

Acheron, a Lupus antigen family member, regulates integrin expression, adhesion, and motility in differentiating myoblasts

Honor L Glenn  1 , Zhaohui Wang, Lawrence M Schwartz
Affiliations

Acheron, a Lupus antigen family member, regulates integrin expression, adhesion, and motility in differentiating myoblasts

Honor L Glenn et al. Am J Physiol Cell Physiol. 2010 Jan.

Abstract

Acheron (Achn) was originally identified as novel gene that is induced when insect muscles become committed to die at the end of metamorphosis. In separate studies, we have demonstrated that Achn acts upstream of MyoD and is required by mammalian myoblasts to either differentiate or undergo apoptosis following loss of growth factors. In the present study we examined the role of Achn in regulating integrin-extracellular matrix interactions that are required for myogenesis. Both control C2C12 myoblasts and those engineered to express ectopic Achn expressed the fibronectin receptor integrin alpha(5)beta(1) in the presence of growth factors and the laminin receptor alpha(7)beta(1) following growth factor withdrawal. Expression of the laminin receptor was blocked in cells expressing either Achn antisense or an Achn deletion mutant that blocks differentiation. Control cells and those expressing ectopic Achn undergo sequential and transient increases in both substrate adhesion and migration before cell fusion. Blockade of Achn expression reduced these effects on laminin but not on fibronectin. Taken together, these data suggest that Achn may influence differentiation in part via its control of cell adhesion dynamics.

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Figures

Fig. 1.
Fig. 1.
Effects of Acheron (Achn) on cell morphology. C2C12 myoblasts were engineered to express an empty vector (pBABE-puro) or one of three Achn variants. A and B: empty vector control. C and D: full-length Achn. E and F: truncated Achn (tAchn). G and H: antisense Achn (AS-Achn). A, C, E, and G: live cells imaged by phase contrast microscopy at ×20 magnification. B, D, F, and H: fixed cells with rhodamine (Rd)-phalloidin to label actin and DAPI to label nuclei. Cells were imaged with fluorescence microscopy at ×40 magnification. Scale bars indicate 50 μm. Insets: ×60 magnification of rhodamine-phalloidin-labeled actin.
Fig. 2.
Fig. 2.
A–C: Western blot analysis of integrin expression in control (Ctrl) and Achn-engineered C2C12 cells. A: β1 (top) and α5 (middle) integrin expression in control, full-length Achn, and truncated Achn expressing cells in growth medium (G) and after 1, 2, or 3 days after transfer to differentiation medium (DM). The constitutively expressed 26S proteasome subunit Trip-1 served as a loading control (bottom). B: α7B integrin expression in control, full-length Achn, truncated Achn, and antisense Achn-engineered cells. C: α7A integrin expression in C2C12 cells. D: Western blot of muscle-specific myosin heavy chain (MHC) in Achn-engineered C2C12 cell lines in growth medium and after 1, 2, and 3 days in DM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.
Fig. 3.
Fig. 3.
RT-PCR analysis of integrin mRNA expression in control and Achn-engineered cells. From top: β1, α5, α6, and α7 in Achn cell lines expressing empty pBABE vector (control), full-length Achn, truncated Achn, and antisense Achn. Cells were analyzed after culture in growth medium (GM) or 1, 2, or 3 days after transfer to DM. GAPDH served as a loading control.
Fig. 4.
Fig. 4.
Effects of Achn on cell adhesion. Percentage of C2C12 cells attached to fibronectin (A) or laminin-1 (B) after culture in GM (DM exposure = 0) and 12, 24, and 48 h after being switched to DM. Control, empty vector. Values are means ± SE of four independent experiments, each performed in triplicate.
Fig. 5.
Fig. 5.
Effects of Achn on the rate of cell spreading on fibronectin. C2C12 cells expressing pBABE-puro vector alone (control), full-length Achn, truncated Achn, or antisense Achn were allowed to spread on fibronectin-coated 35-mm dishes as described. A: cells maintained in GM. B: cells in DM for 12 h. C: cells in DM for 24 h. D: cells in DM for 48 h. Values are means ± SE of three independent experiments, each performed in duplicate.
Fig. 6.
Fig. 6.
Effects of Achn on the rate of cell spreading on laminin. C2C12 cells expressing pBABE-puro vector alone (control), full-length Achn, truncated Achn, or antisense Achn were allowed to spread on laminin-coated 35-mm dishes. A: cells maintained in GM. B: cells in DM for 12 h. C: cells in DM for 24 h. D: cells in DM for 48 h. Values are means ± SE of three independent experiments, each performed in duplicate.
Fig. 7.
Fig. 7.
Effects of Achn on cell motility during differentiation. C2C12 cells expressing pBABE-puro vector alone (Con), full-length Achn, truncated Achn, or antisense Achn were allowed to attach and migrate through fibronectin-coated (A) or laminin-1-coated (B) 8-μm filters. Cells were assayed in GM (DM exposure = 0) and after 12, 24, and 48 h after transfer to DM. The fraction of cells that migrated through the filter relative to total attached cells was calculated and then normalized to the control values in GM. Values are means ± SE of four independent experiments, each performed in triplicate.
Fig. 8.
Fig. 8.
A: model of adhesion events during early myoblast differentiation. Our data support a model in which cells undergo an adhesion phase that peaks at 12 h following loss of growth factors (dashed line). The resolution of this phase coincides with the initiation of a migratory phase that peaks at 24 h (solid line). Migration in turn declines as cells initiate fusion (dotted line), which results in the conversion of mononucleated myoblasts into multinucleated myotubes. B: expression of truncated Achn alters adhesion dynamics of myoblasts on a laminin substrate. Adhesion increases at 12 h after cells are stimulated to differentiate but remains elevated (dashed line). Both migration (solid line) and fusion (dotted line) are inhibited. C: expression of antisense Achn blocks adhesion (dashed line) as well as migration (solid line) on laminin and also inhibits fusion.
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