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

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.

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.

Figure 2

Immunohistochemical detection of LM α2 chain in round and elongated cells in monocultures and in organotypic epithelial-mesenchymal cocultures. (A) mouse mesenchymal cells cultured for 24 h in 0.05% poly-l-lysine coated dishes remain round and negative for LM α2 chain. (B) Mesenchymal cells plated at high cell density remain negative for LM α2 chain after 24 h in culture. (C) Mesenchymal cells allowed to spread for 24 h synthesize LM α2 chain (red). (D) Epithelial-mesenchymal organotypic cocultures. Mesenchymal cells spread around epithelial cell spheres (e) and express LM α2 (arrow). The rest of the mesenchymal cells (m) form a monolayer of round cells and remain negative for LM α2. (Inset) Organotypic cultures after three days have well developed epithelial cysts with central lumens. More mesenchymal cells spread around the cysts and become positive for LM α2 chain. Bars: (A–D) 15 μm; (inset) 60 μm.

Figure 3

LM α2 chain in round and elongated cells of the embryonic lung. Western blot using a polyclonal antibody to LM-1/LM-2. Lane 1 (lung R), day 10.5 lungs, containing only round cells. Lane 2 (lung E), airways and surrounding mesenchyme microdissected from day 12 lungs, containing mainly elongated cells. mes. (lane 3), peripheral mesenchyme isolated from E12 lungs, containing only round cells (R). Lane 4 (R1), round undifferentiated mesenchymal cells before culture. Lane 5 (R2), round mesenchymal cells plated at high density to prevent cell spreading. LM α2 chain is seen only in elongated cells (lane 2).

Figure 4

Switching cells from culture conditions promoting rounding or elongation. Mesenchymal cells cultured on poly-l-lysine (R) or with poly-l-lysine added to the culture medium after cell spreading is completed (E). After 24 h the cells were replated and cultured for an additional 24 h under similar or opposite conditions. Then immunoblotting was performed using a polyclonal antibody to LM-1/LM-2. LM α2 band appears in elongated cells (second and third lane) and is absent in the round cells (fourth and fifth lane). The first lane is LM-1 from EHS tumor, used as control.

Figure 5

LM α2 chain expression in mesenchymal cells from other organs and in lung epithelial cells. Western blot using a polyclonal antibody to LM-1/LM-2 on mesenchymal cells from day 15 intestine and day 12 kidney, and epithelial cells from day 15 lungs cultured on poly-l-lysine (R) or with poly-l-lysine added to the culture medium after cell spreading is completed (E). Note the presence of LM α2 band in elongated cells (E) from all three sources. The other lanes show β1 and γ1 bands only. The first lane is LM-1 from the EHS tumor, used as control.

Figure 6

Cell spreading perturbation assays using antibodies to LM α2 chain. Lung mesenchymal cells were cultured in the presence of 1 and 2 μg/ml of LM α2 antibody, IgG control, or 2 μg/ml of LM α2 antibody preincubated with LM-2. Cell spreading was measured after 24 h in culture. (A) Cells in the presence of 2 μg/ml IgG. (B) Cells in the presence of 2 μg/ml LM α2 antibody. (C) Histogram obtained by measuring the maximal cell diameters under the various treatments. The inhibition in cell spreading was statistically significant, P < 0.0001 for 2 μg/ml LM α2 antibody. (D) Immunoblot showing less SM α actin in cells exposed to the anti-LM α2 chain antibody. Bar, 40 μm.

Figure 7

SM α actin production by lung mesenchymal cells isolated from adult dy/dy mice and controls. Cell spreading and SM α actin production were determined after 24 h in culture. The histogram shows the maximal cell diameters of wild-type and dy/dy mouse cells cultured on LM-1, LM-2, or uncoated dishes. The immunoblot shows SM α actin production by the cells. Notice the short diameter and low level of SM α actin in dy/dy mouse cells when cultured on LM-1 or uncoated dishes but not when cultured on LM-2.

Figure 8

SM cell size in intraparenchymal bronchi of dy/dy mice and controls. (A) Histogram showing maximal nuclear diameter as an indicator of cell size in hematoxylin eosin-stained histological sections of lungs (cell boundaries are not seen in organs). Dy/dy mice showed shorter nuclei compared with controls (P < 0.05). (B) Immunoblots demonstrating lower levels of SM proteins in dy/dy lungs compared with controls.

Figure 9

SM in trachea and bronchi of dy/dy mice and controls. (A–D) Hematoxylin eosin-stained histological sections of trachea and main bronchi. (A) Normal tracheal SM (arrows) surrounding tracheal cartilage; compare to the severely underdeveloped tracheal SM in dy/dy mouse, shown in B (arrows). (C) SM in normal main bronchus (arrow) is significantly thicker than in dy/dy mouse (D). (E and F) intraparenchymal bronchial SM immunostained with antibodies to SM α actin (brown). (E) Normal SM (arrow) surrounding medium sized bronchi; compare to thinner, more discontinuous bronchial muscle in dy/dy mice, shown in F (arrow). Bars: (A–D) 20 μm; (E and F) 60 μm.

Figure 10

SM cell size in circular layer of intestinal muscularis propria of dy/dy mice and controls. (A) Histogram showing maximal nuclear diameter as an indicator of cell size in hematoxylin eosin-stained histological sections (cell boundaries are not seen in organs). Dy/dy mice showed shorter nuclei compared with controls (P < 0.03). (B) Immunoblots demonstrating slightly higher levels of SM proteins in dy/dy intestine compared with controls, consistent with the hyperplasia described in Results.

Figure 11

Proposed role of LM-1 and LM-2 in bronchial myogenesis. e, Epithelial cells (gray); m, mesenchymal cells (yellow); SM, smooth muscle cells (from pink to red); BM, basement membrane. The asterisk tags the relative position adopted by an epithelial cell from the moment it is born and establishes a new contact with the mesenchyme.