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Cell elongation induces laminin alpha2 chain expression in mouse embryonic mesenchymal cells: role in visceral myogenesis.

Relan NK, Yang Y, Beqaj S, Miner JH, Schuger L - J. Cell Biol. (1999)

Bottom Line: In comparison, the expression of LM beta1 and gamma1 remains unchanged.These deficiencies were completely corrected by exogenous LM-2.The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

ABSTRACT
Bronchial smooth muscle (SM) mesenchymal cell precursors change their shape from round to spread/elongated while undergoing differentiation. Here we show that this change in cell shape induces the expression of laminin (LM) alpha2 chain not present in round mesenchymal cells. LM alpha2 expression is reversible and switched on and off by altering the cell's shape in culture. In comparison, the expression of LM beta1 and gamma1 remains unchanged. Functional studies showed that mesenchymal cell spreading and further differentiation into SM are inhibited by an antibody against LM alpha2. Dy/dy mice express very low levels of LM alpha2 and exhibit congenital muscular dystrophy. Lung SM cells isolated from adult dy/dy mice spread defectively and synthesized less SM alpha-actin, desmin, and SM-myosin than controls. These deficiencies were completely corrected by exogenous LM-2. On histological examination, dy/dy mouse airways and gastrointestinal tract had shorter SM cells, and lungs from dy/dy mice contained less SM-specific protein. The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity. This study therefore demonstrated a novel role for the LM alpha2 chain in SM myogenesis and showed that its decrease in dy/dy mice results in abnormal SM.

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LM α2 chain expression in round versus elongated lung mesenchymal cells. (A) Western blot using a polyclonal antibody to LM-1/LM-2 on cells cultured for 4 and 24 h on 10- (R, round) or 20-μm microsurfaces (E, elongated). Note the presence of LM α2 band in elongated cells (E) after 24 h of culture. LM α2 also appears in the culture media after 24 h of culture. The first lane is LM-1 from the EHS tumor, used as control. (B) Cell lysates from round (R) and elongated (E) mesenchymal cells were immunoprecipitated with a monoclonal antibody against LM α2 chain and blotted with a polyclonal antibody to LM-1/LM-2. The immunoblot shows that the elongated cells but not the round cells synthesize trimeric LM-2. (C) RT-PCR showing higher levels of LM α2 chain mRNA in elongated (E), compared with round cells (R) (30 PCR cycles for α1 and β1 chains, 25 cycles for β1 and γ1 chains). (D) RPA confirming the higher level of LM α2 chain mRNA in elongated (E) cells.
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Figure 1: LM α2 chain expression in round versus elongated lung mesenchymal cells. (A) Western blot using a polyclonal antibody to LM-1/LM-2 on cells cultured for 4 and 24 h on 10- (R, round) or 20-μm microsurfaces (E, elongated). Note the presence of LM α2 band in elongated cells (E) after 24 h of culture. LM α2 also appears in the culture media after 24 h of culture. The first lane is LM-1 from the EHS tumor, used as control. (B) Cell lysates from round (R) and elongated (E) mesenchymal cells were immunoprecipitated with a monoclonal antibody against LM α2 chain and blotted with a polyclonal antibody to LM-1/LM-2. The immunoblot shows that the elongated cells but not the round cells synthesize trimeric LM-2. (C) RT-PCR showing higher levels of LM α2 chain mRNA in elongated (E), compared with round cells (R) (30 PCR cycles for α1 and β1 chains, 25 cycles for β1 and γ1 chains). (D) RPA confirming the higher level of LM α2 chain mRNA in elongated (E) cells.

Mentions: LM α2 chain expression is prevented by cell rounding and is induced by cell spreading/elongation. Regardless of the culture system used to facilitate cell rounding or cell spreading, we consistently observed that only spread cells expressed LM α2 chain. Immunoblots of round and elongated lung mesenchymal cells plated on culture microsurfaces showed LM α2 chain as a protein with a Mr of ∼350 kD present only in lysates from elongated cells (Fig. 1 A). The band was not detected during the first 4 h in culture, but progressively appeared within the next 20 h. Unlike the LM α2 chain, no modulations were found in expression of the LM β1 and γ1 chains, which was similar for both round and elongated cells. We used a monospecific antibody to confirm the identity of LM α2 chain. Since this antibody is not suited for immunoblotting, we used it for immunoprecipitation, followed by immunoblotting with the anti–LM-1/LM-2 antibody mentioned above. The lack of LM-2 in the round cell immunoprecipitates confirmed that only elongated cells synthesize LM α2 (Fig. 1 B).


Cell elongation induces laminin alpha2 chain expression in mouse embryonic mesenchymal cells: role in visceral myogenesis.

Relan NK, Yang Y, Beqaj S, Miner JH, Schuger L - J. Cell Biol. (1999)

LM α2 chain expression in round versus elongated lung mesenchymal cells. (A) Western blot using a polyclonal antibody to LM-1/LM-2 on cells cultured for 4 and 24 h on 10- (R, round) or 20-μm microsurfaces (E, elongated). Note the presence of LM α2 band in elongated cells (E) after 24 h of culture. LM α2 also appears in the culture media after 24 h of culture. The first lane is LM-1 from the EHS tumor, used as control. (B) Cell lysates from round (R) and elongated (E) mesenchymal cells were immunoprecipitated with a monoclonal antibody against LM α2 chain and blotted with a polyclonal antibody to LM-1/LM-2. The immunoblot shows that the elongated cells but not the round cells synthesize trimeric LM-2. (C) RT-PCR showing higher levels of LM α2 chain mRNA in elongated (E), compared with round cells (R) (30 PCR cycles for α1 and β1 chains, 25 cycles for β1 and γ1 chains). (D) RPA confirming the higher level of LM α2 chain mRNA in elongated (E) cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2168094&req=5

Figure 1: LM α2 chain expression in round versus elongated lung mesenchymal cells. (A) Western blot using a polyclonal antibody to LM-1/LM-2 on cells cultured for 4 and 24 h on 10- (R, round) or 20-μm microsurfaces (E, elongated). Note the presence of LM α2 band in elongated cells (E) after 24 h of culture. LM α2 also appears in the culture media after 24 h of culture. The first lane is LM-1 from the EHS tumor, used as control. (B) Cell lysates from round (R) and elongated (E) mesenchymal cells were immunoprecipitated with a monoclonal antibody against LM α2 chain and blotted with a polyclonal antibody to LM-1/LM-2. The immunoblot shows that the elongated cells but not the round cells synthesize trimeric LM-2. (C) RT-PCR showing higher levels of LM α2 chain mRNA in elongated (E), compared with round cells (R) (30 PCR cycles for α1 and β1 chains, 25 cycles for β1 and γ1 chains). (D) RPA confirming the higher level of LM α2 chain mRNA in elongated (E) cells.
Mentions: LM α2 chain expression is prevented by cell rounding and is induced by cell spreading/elongation. Regardless of the culture system used to facilitate cell rounding or cell spreading, we consistently observed that only spread cells expressed LM α2 chain. Immunoblots of round and elongated lung mesenchymal cells plated on culture microsurfaces showed LM α2 chain as a protein with a Mr of ∼350 kD present only in lysates from elongated cells (Fig. 1 A). The band was not detected during the first 4 h in culture, but progressively appeared within the next 20 h. Unlike the LM α2 chain, no modulations were found in expression of the LM β1 and γ1 chains, which was similar for both round and elongated cells. We used a monospecific antibody to confirm the identity of LM α2 chain. Since this antibody is not suited for immunoblotting, we used it for immunoprecipitation, followed by immunoblotting with the anti–LM-1/LM-2 antibody mentioned above. The lack of LM-2 in the round cell immunoprecipitates confirmed that only elongated cells synthesize LM α2 (Fig. 1 B).

Bottom Line: In comparison, the expression of LM beta1 and gamma1 remains unchanged.These deficiencies were completely corrected by exogenous LM-2.The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

ABSTRACT
Bronchial smooth muscle (SM) mesenchymal cell precursors change their shape from round to spread/elongated while undergoing differentiation. Here we show that this change in cell shape induces the expression of laminin (LM) alpha2 chain not present in round mesenchymal cells. LM alpha2 expression is reversible and switched on and off by altering the cell's shape in culture. In comparison, the expression of LM beta1 and gamma1 remains unchanged. Functional studies showed that mesenchymal cell spreading and further differentiation into SM are inhibited by an antibody against LM alpha2. Dy/dy mice express very low levels of LM alpha2 and exhibit congenital muscular dystrophy. Lung SM cells isolated from adult dy/dy mice spread defectively and synthesized less SM alpha-actin, desmin, and SM-myosin than controls. These deficiencies were completely corrected by exogenous LM-2. On histological examination, dy/dy mouse airways and gastrointestinal tract had shorter SM cells, and lungs from dy/dy mice contained less SM-specific protein. The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity. This study therefore demonstrated a novel role for the LM alpha2 chain in SM myogenesis and showed that its decrease in dy/dy mice results in abnormal SM.

Show MeSH
Related in: MedlinePlus