ADAM12 and alpha9beta1 integrin are instrumental in human myogenic cell differentiation

Abstract

Knowledge on molecular systems involved in myogenic precursor cell (mpc) fusion into myotubes is fragmentary. Previous studies have implicated the a disintegrin and metalloproteinase (ADAM) family in most mammalian cell fusion processes. ADAM12 is likely involved in fusion of murine mpc and human rhabdomyosarcoma cells, but it requires yet unknown molecular partners to launch myogenic cell fusion. ADAM12 was shown able to mediate cell-to-cell attachment through binding alpha9beta1 integrin. We report that normal human mpc express both ADAM12 and alpha9beta1 integrin during their differentiation. Expression of alpha9 parallels that of ADAM12 and culminates at time of fusion. alpha9 and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/alpha9beta1 integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to alpha9beta1, inhibited overall mpc fusion by 47-48%, with combination of both strategies increasing inhibition up to 62%. By contrast with blockade of vascular cell adhesion molecule-1/alpha4beta1, which also reduced fusion, exposure to ADAM12 antisense oligonucleotides or anti-alpha9beta1 antibody did not induce detachment of mpc from extracellular matrix, suggesting specific involvement of ADAM12-alpha9beta1 interaction in the fusion process. Evaluation of the fusion rate with regard to the size of myotubes showed that both ADAM12 antisense oligonucleotides and alpha9beta1 blockade inhibited more importantly formation of large (> or =5 nuclei) myotubes than that of small (2-4 nuclei) myotubes. We conclude that both ADAM12 and alpha9beta1 integrin are expressed during postnatal human myogenic differentiation and that their interaction is mainly operative in nascent myotube growth.

Figure 1.

In vitro differentiation of human mpc. (A) mpc growth is expressed in number of cells per square centimeter (closed circles), and mpc differentiation is estimated by the fusion index (open circles). There is no significant increase of fusion index from day 14 to day 21. Results are means ± SD of eight independent cultures. (B) Myogenin immunoblot at days 4, 7, and 14 of mpc culture. (C) May-Grünwald Giemsa stain of mpc at day 4 (d4), 7 (d7), and 14 (d14) of culture. Bar, 40 μm.

Figure 2.

ADAM12 expression by human mpc. (A) RT-PCR analysis of ADAM12-L, ADAM12-S, and β2-microglobulin mRNAs in mpc at days 4, 7, and 14 of culture (expected sizes of PCR products were 314, 371, and 335 base pairs, respectively). (B) Immunoblot analysis of pro-(100-kDa) and mature (90-kDa) ADAM12-L (top) in mpc lysates with goat polyclonal anti-cytoplasmic tail of ADAM12-L, and of pro-(92-kDa) and mature (68-kDa) ADAM12-S (bottom) in mpc supernatants with mouse monoclonal 2F7 directed toward disintegrin domain of ADAM12, at day 4, 7, and 14. (C) ADAM12 localization (FITC) in Triton X-100 permeabilized mpc by using rb122 antibody. (D) ADAM12 localization (FITC) in unpermeabilized mpc. (E) After permeabilization, differentiated mpc (day 7) were labeled with rb122 antibody revealed by FITC-conjugated secondary antibodies (green) and with myogenin antibody revealed by TRITC-conjugated secondary antibodies (red). Nuclei staining with DAPI. Bars, 20 μm.

Figure 3.

α9β1 expression by human mpc. (A) α₉ and b₁ integrin subunits in mpc at days 4, 7, and 14 of culture, analyzed by RT-PCR. Expected size of PCR products was, respectively, 424 and 881 base pairs. (B) Detection by immunoblot analysis of mature α₉ (150 kDa) in cultured cell lysate at days 4, 7, and 14. (C) Unpermeabilized mpc exhibited membrane immunolabeling of α₉β₁ integrin with monoclonal Y9A2 antibody (directed toward α₉β₁) by using FITC-conjugated secondary antibody. Propidium iodide stain of nuclei. Bars, 20 μm.

Figure 4.

Membrane expression of ADAM12 and α₉β₁ integrin and colocalization at cell-cell contact. Double immunolabeling on unpermeabilized mpc of ADAM12 (rb122) and α₉β₁ integrin (Y9A2) by using TRITC- or FITC-conjugated secondary antibodies, respectively. DAPI stain of nuclei. Bar, 20 μm.

Figure 6.

α₉β₁ integrin acts as a ligand for mpc membrane-bound ADAM12. (A) Immunoblot analysis of ADAM12 expression (with rb122, anti-disintegrin domain) after coimmunoprecipitation with irrelevant mouse immunoglobulins or monoclonal anti-Y9A2 antibody (left), and of α₉ expression (with #1057, anti-cytoplasmic domain) after coimmunoprecipitation with irrelevant mouse immunoglobulins or monoclonal anti-ADAM12 antibodies (6E6, 8F8, and 6C10 mix) (right). Example of one blot. (B) Percentage of bound cells to plate coated with purified full length ADAM12 and incubated with either irrelevant mouse Ig, anti-α₉β₁ (Y9A2), or anti-a₇b₁ (O26) blocking antibodies. Results are means ± SD of three experiments run in triplicate (p < 0.05).

Figure 5.

ADAM12 and α₉β₁ integrin in mpc fusion. (A) Immunoblot quantification of ADAM12 isoforms expression by mpc-treated or not with an ADAM12 antisense and nonsense. Graph shows densitometric analysis of three different cultures, and immunoblot presents an example of one culture. (B) May-Grünwald Giemsa stain of mpc at day 14 showing morphological appearance of myotubes after different treatments. Bar, 40 μm. (C) Fusion index was measured for each situation and normalized as percentage of the control (untreated cells). Results are means ± SD of three experiments run in duplicate (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). (D) Percentage of nuclei included in mononucleated cells, in cells with two to four nuclei and in cells with five or more nuclei under the total number of nuclei (^**p < 0.01, ^***p < 0.001). (E) Percentage of nuclei included in myotubes with two to four nuclei (left) or five or more nuclei (right) under the total number of nuclei in myotubes with two or more nuclei (^*p < 0.05, ^***p < 0.01, ^***p < 0.001).

Figure 7.

α₉β₁ integrin binding to ADAM12 induces intercellular mpc adhesion. (A) Cell deadhesion was measured for each situation and normalized as percentage of the control (untreated cells). Results are means ± SD of three experiments run in triplicate (^*p < 0.03). (B) Adhesion of cells treated or not with nonsense or antisense oligonucleotides on untreated cell layer after incubation with mouse immunoglobulins, anti-α₉β₁ (Y9A2), or anti-a₇b₁ (O26) blocking antibodies. (C) Adhesion of cells treated or not with nonsense or antisense oligonucleotides on nonsense treated cell layer after incubation with mouse immunoglobulins, anti-α₉β₁ (Y9A2), or anti-α₇β₁ (O26) blocking antibodies. (D) Adhesion of cells treated or not with nonsense or antisense oligonucleotides on antisense treated cell layer after incubation with mouse immunoglobulins, anti-α₉β₁ (Y9A2), or anti-a₇b₁ (O26) blocking antibodies. Results are means ± SD of three experiments run in triplicate.