Correspondence to: Lucia Schuger, Department of Pathology, Wayne State University, 540 E. Canfield, Detroit, MI 48201. Tel:(313) 577-5651 Fax:(313) 577-0057 E-mail:lschuger{at}med.wayne.edu.
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Abstract |
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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) 2 chain not present in round mesenchymal cells. LM
2 expression is reversible and switched on and off by altering the cell's shape in culture. In comparison, the expression of LM ß1 and
1 remains unchanged. Functional studies showed that mesenchymal cell spreading and further differentiation into SM are inhibited by an antibody against LM
2. Dy/dy mice express very low levels of LM
2 and exhibit congenital muscular dystrophy. Lung SM cells isolated from adult dy/dy mice spread defectively and synthesized less SM
-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
2 chain in SM myogenesis and showed that its decrease in dy/dy mice results in abnormal SM.
Key Words: laminin, smooth muscle, myogenesis, dy/dy mice, lung
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Introduction |
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LAMININS (LMs)1 are heterotrimeric basement membrane glycoproteins composed of , ß and
chains linked together by disulfide bonds in a cruciform tertiary structure. The first LM identified, referred to as LM-1, is composed of
1, ß1, and
1 chains (
2, ß1, and
1 chains (
2 chain binds covalently to LM ß2 and
1 chains to form LM-4 (
The human LM 2 chain gene (Lama2) was localized to chromosome 6q22-23 (
2 chain gene cause autosomal recessive congenital muscular dystrophies in humans and in dy/dy mice (
2 chain (
2 chain gene in mice by homologous recombination has confirmed its importance in skeletal muscle development and function (
The laminin 2 chain is expressed in the developing and adult human and mouse lung (
1 chain and coincides with the period of active bronchial myogenesis (
2 chain in the developing lung has not been elucidated, although it has been shown to facilitate attachment of lung myofibroblasts in culture (
During development, embryonic cells undergo significant changes in shape, starting as round pluripotent cells and culminating in the multiple cellular configurations seen in mature tissues. Our studies (1 chain deposition and further polymerization at the airway basement membrane site (
Here we show that cell spreading/elongation, whether in vivo or in vitro, activates expression of the LM 2 chain, which is absent in round cells. In addition, by blocking LM
2 with a specific antibody, we demonstrate that, once secreted, LM
2 promotes mesenchymal cell spreading/elongation and further SM myogenic differentiation. Our findings thus provide a potential explanation for how SM myogenesis could proceed after LM-1 in the epithelial basement membrane stimulates the first layer of mesenchymal cells to elongate and differentiate.
Since dy/dy mice express low levels of LM 2 chain, we used their cells to further study the role of this LM chain in myogenesis. Here we show that lung mesenchymal cells isolated from dy/dy mice spread defectively in culture and synthesize less SM
-actin, desmin and SM myosin than controls. These abnormalities were completely overcome by the addition of exogenous LM-2. Furthermore, histological examination of dy/dy mouse tissues revealed subtle but diffuse SM cell shortening that, in the absence of compensatory muscle hyperplasia, resulted in lower levels of SM-specific proteins in immunoblots. Our studies therefore demonstrate a novel role for LM
2 chain in visceral myogenesis. Its regulation by cell shape may contribute to a multilayered bronchial SM, and its absence may cause abnormal visceral muscle.
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Materials and Methods |
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LMs and Antibodies
LM-1 from the mouse EHS tumor, type IV collagen, and fibronectin were purchased from Becton-Dickinson, and LM-2 was obtained from GIBCO BRL. A polyclonal rat antibody to EHS LM which recognizes LM 1,
2, and ß1/
1 chains (LM-1/LM-2) (
2 chain (
-actin was obtained from Boehringer Mannheim, rabbit polyclonal antibodies to SM myosin were purchased from Biomedical Technologies, and a mouse monoclonal antibody to desmin was purchased from Dako (Carpinteria, CA). HRP-labeled secondary antibodies were purchased from Bio-Rad and normal rat IgG was purchased from Cappel.
Cells and Lung Fragments
To obtain embryonic mesenchymal cells, Crl: CD-1® (ICR) BR mice (Charles River) were mated and the day of finding a vaginal plug was designated as day zero of embryonic development. Lungs were removed at days 1215 of gestation, minced, and then trypzinised into a single cell suspension, and the mesenchymal cells were isolated by differential plating as previously described (
In some studies, tissues were isolated from the embryo, immediately lysed, and used for immunoblot analysis. Whole lungs were microdissected from day 1010.5 mouse embryos. Fragments of undifferentiated, round mesenchymal cells were microdissected from the periphery of day 12 mouse embryonic lungs, and fragments of elongated mesenchymal cells were microdissected, together with epithelial cells, from the proximal/central part of day 12 lungs.
Immunoblotting and Immunoprecipitation
Cells and tissues from CD-1, C57BL/6J dy/dy mice and C57BL/6J +/+ normal mice were lysed and resolved under reducing conditions by PAGE. Immunoblotting for LMs and SM proteins was then done as described (2 chain for 1624 h at 4°C in a rotary shaker. The immune complex was precipitated with protein Aconjugated Sepharose and resolved by 3.5% PAGE. The samples were transferred to nitrocellulose membranes and blotted with polyclonal antibody to LM-1/LM-2 (1:100 dilution) and bands were detected by chemiluminescence as previously described (
RT-PCR and Ribonuclease Protection Assay
Mesenchymal cell cultures were briefly washed with PBS, lysed with Trizol (GIBCO BRL) and total RNA was extracted and used for RT-PCR and ribonuclease protection assay (RPA). RT-PCR was performed with the GeneAmp® RNA PCR kit (Perkin Elmer) following the manufacturer's instructions. 25 cycles were run for amplification of ß1 and 1 chains and 30 for amplification of
1,
2 chains,
4 and
5 chains. The following primers were used for PCR: LM
1 chain 5' forward primer: 5'-tgggtgtgggatttcttagc-3' and 3' reverse primer: 5'-cctgaccgtctacccagtgt-3', LM
2 chain 5' forward primer: 5'-gcctgccaactctgagaaac-3' and 3' reverse primer: 5'-tcccaagagaatgatcccag-3', LM
4 chain 5' forward primer: 5'-gccatcgaagaagtagctgg-3' and 3'-reverse primer: 5'-cccctgaggacactgtgttt-3', LM
5 chain 5' forward primer: 5'-tgaaggcagagggtagcagt-3' and 3' reverse primer 5'-gttgtctgcctggcttcttc-3', LM ß1 chain 5' forward primer: 5'-gttcgagggaactgcttctg-3' and 3' reverse primer: 5'-tgccagtagccaggaagact-3'. LM
1 chain 5' forward primer: 5'-gagctttgaggacgaccttg-3' and 3' reverse primer 5'-ctggggtgtgtatgctgatg-3'.
For RPA a 356-bp LM 2 chain fragment which was cloned in the pCR II vector (
-[32P] (400900 Ci/mmol and 10 mCi/ml; NEN). The hybridization was carried out using an RPA kit (Ambion Inc.) and following the manufacturer's instructions. A 256-bp GAPDH linearized fragment obtained from Ambion Inc. was used as a control. The protected RNA fragments were resolved on a 5% denaturing polyacrylamide gel and autoradiographed.
Cell Attachment and Spreading Assays
Freshly isolated lung mesenchymal cells were cultured in the presence of anti-LM 2 chain antibody, anti-LM
2 chain antibody preincubated with 10-fold excess of LM-2 (molar:molar) or IgG control. The antibody and IgG were added at concentrations of 1, 2, 5, and 10 µg/ml to the cultures either at the time of cell plating to determine their effect on cell attachment or 40 min after plating, when attachment was completed, to determine their effect on cell spreading. The cultures were maintained for 2 and 4 h for cell attachment studies and for 4 and 24 h for cell spreading studies. At the end of the culture period the cells were either fixed or lysed. Fixed cells were counted and their diameters were determined on images projected on a computer screen with the aid of a digitized pad and the image analysis program Optimas 5.1. (
actin, anti-desmin and anti-SM myosin antibodies as described above. In additional experiments mesenchymal cells were isolated from adult C57BL/6J dy/dy and C57BL/6J +/+ normal mice and plated on culture dishes uncoated or coated with 10 µg/ml of LM-2 as indicated in the manufacturer's instructions for cell attachment assays or with the same concentration of LM-1 or fibronectin. Cell attachment and spreading was then determined as just described.
Histological Studies
Lungs with trachea, stomach, and intestine from adult C57BL/6J dy/dy, C57BL/6J +/+ normal mice and CD-1 mice were formalin-fixed and paraffin-embedded. 5-µm sections were mounted on glass slides and stained with hematoxilin-eosin for light microscopic examination. Since the nuclei of SM cells are proportional to the cell's length and the cell boundaries are indistinct in vivo, nuclear length was determined as an indicator of cell elongation. Up to 460 nuclei (lung, 216; stomach, 120; intestine, 124 nuclei) were measured as described above in sections from lung, stomach, and intestine dissected from four dy/dy, four C57BL/6J +/+ and two CD-1 mice.
Immunohistochemistry
Mesenchymal cell cultures and organotypic epithelial-mesenchymal cocultures were immunostained with anti-LM 2 antibodies and frozen sections from dy/dy and normal lungs were immunostained with anti-SM
-actin following previous protocols (
2 and diaminobenzidine tetrahydrochloride (DAB) (brown color) was used for detecting SM
actin.
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Results |
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LM 2 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
2 chain. Immunoblots of round and elongated lung mesenchymal cells plated on culture microsurfaces showed LM
2 chain as a protein with a Mr of ~350 kD present only in lysates from elongated cells (Figure 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
2 chain, no modulations were found in expression of the LM ß1 and
1 chains, which was similar for both round and elongated cells. We used a monospecific antibody to confirm the identity of LM
2 chain. Since this antibody is not suited for immunoblotting, we used it for immunoprecipitation, followed by immunoblotting with the antiLM-1/LM-2 antibody mentioned above. The lack of LM-2 in the round cell immunoprecipitates confirmed that only elongated cells synthesize LM
2 (Figure 1 B).
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RT-PCR and RPAs demonstrated that the increment in LM 2 chain is, at least in part, the result of an increment in its steady-state mRNA levels (Figure 1 C). RT-PCR demonstrated no changes in mRNA levels for LM
1, ß1, and
1 chains between round and elongated cells as well as no changes in LM
4 and
5 message levels (not shown). As expected, LM
1 polypeptide chain was not detected under any of these culture conditions. Our previous studies showed that LM
1 chain synthesis requires epithelial-mesenchymal cellcell contact (
2 chain synthesis between cells plated on uncoated dishes or dishes coated with LM-1, LM-2, collagen IV, and fibronectin (not shown).
Immunohistochemical studies confirmed the absence of LM 2 chain in round mesenchymal cells and its presence in elongated cells (Figure 2, AC). In organotypic cultures epithelial cells rearrange into spheres and mesenchymal cells surround them forming also a surface-anchored monolayer (
2, whereas the rest remained negative (Figure 2 D). Over time the mesenchymal cells formed cysts with central lumens and additional mesenchymal cells spread/elongated around them and synthesized LM
2 chain (Figure 2, inset).
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In vivo undifferentiated (round) mesenchymal cells do not express LM 2 chain, but elongated cells do. Here we determined whether the observations made in tissue cultures were relevant to the lung in vivo. At day 10.5 of gestation all the cells in the lung are round in shape. At this stage, the embryonic lung synthesizes LM
1 but not LM
2 (Figure 3, first lane). At day 12 of gestation, most of the proximal peribronchial mesenchymal cells become spread/elongated and the distal epithelial cells become spread/flat. The undifferentiated mesenchymal cells, however, are still round. Western blots of microdissected airways and surrounding mesenchyme showed that the elongated cells express both LM
1 and
2 chains (Figure 3, second lane), whereas Western blots of microdissected undifferentiated mesenchyme showed absence of LM
2 chains in round cells (Figure 3, third lane).
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LM 2 chain expression is turned on and off by changes in cell shape. Switching cells from culture conditions that promote cell rounding to culture conditions that promote cell elongation and vice versa demonstrated that synthesis of LM
2 chain can be switched on and off by changes in cell shape (Figure 4). Replating efficiency was similar for round and elongated cells (48 ± 12 and 50 ± 10% of replated cells attached, respectively).
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The control of LM 2 chain expression by the cell shape is not restricted to lung embryonic mesenchymal cells. During lung development, both epithelial and mesenchymal cells undergo changes in shape. The most dramatic changes take place on distal epithelial cells that become totally flat and spread by day 1415 of gestation. Here we determined the role of cell shape on lung embryonic epithelial cells as well as on embryonic mesenchymal cells from kidney and intestine. Immunoblots showed that cell shape also regulates LM
2 chain expression in these cells in a similar manner as observed in mesenchymal cells. Like in embryonic mesenchymal cells, rounding prevented and spreading induced LM
2 chain synthesis (Figure 5).
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Cell spreading is blocked by monoclonal antibodies to LM 2 chain. Effect on SM differentiation. In functional studies, 1 and 2 µg /ml of monoclonal antibody against LM
2 chain were sufficient to block mesenchymal cell spreading in a statistically significant manner (Figure 6, AC). Higher concentrations of antibody did not further reduce spreading. Therefore, these studies indicated a reciprocal interaction between cell shape and LM
2 by showing that not only is the LM
2 chain induced by cell spreading, but also that cell spreading is induced by the LM
2 chain. Mesenchymal cells exposed to the anti-LM
2 chain antibodies showed a decrease in synthesis of SM-specific proteins, including SM
-actin, desmin and myosin when compared with cells exposed to control immunoglobulin (Figure 6 D, and data not shown). Attachment assays in the presence of anti-LM
2 chain antibody or IgG control showed that the antibody also inhibited cell adhesion (64% less cells attached in the presence of 20 µg/ml of anti-LM
2 chain antibodies than in the presence of same concentration of control IgG; not shown).
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SM cells from adult dy/dy mouse lung spread poorly in culture and contain less SM-specific protein. Over 40% of the attached mesenchymal cells, whether isolated from normal or mutant mice, reacted with anti-desmin antibodies (not shown). All mesenchymal cell cultures isolated from adult dy/dy mouse lungs showed poor cell spreading and lower levels of SM-specific protein than controls. The deficiencies in cell spreading and SM protein synthesis disappeared by plating the cells on LM-2 (Figure 7). LM-1 or fibronectin coating did not improve the abnormalities in cell spreading and SM protein synthesis seen in dy/dy mouse cells (Figure 7 and data not shown).
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Dy/dy mice have SM abnormalities in vivo, including shorter cells and deficiencies in SM-specific protein production. Cell morphometry studies showed that bronchial SM cells in dy/dy mice were shorter than in control animals in a statistically significant manner (Figure 8 A). No differences in size were found, however, in lung vascular SM cells or bronchial columnar epithelial cells (not shown). Immunoblots demonstrated that adult dy/dy mouse lungs contained less SM-specific proteins, including SM -actin, desmin, and myosin (Figure 8 B). On histological examination, the most abnormal SM was that of the trachea and main bronchi. In those sites, the visceral SM cells were shorter and underdeveloped (Figure 9B and Figure D). For comparison, A and C show tracheal and bronchial SM in normal animals of the same strain. Unlike the extraparenchymal airway, the changes in the intraparenchymal bronchial SM were subtle and detected mainly by cell morphometry (shorter cells) and immunoblotting as already shown in Figure 8. On immunohistochemistry using an anti-SM
-actin antibody, the large and medium sized bronchial muscle exhibited slightly, but clear thinning and more discontinuity than the normal counterparts (Figure 9E and Figure F).
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The SMs of stomach and intestine were also studied. Histological examination and morphometric studies of the stomach revealed no alterations in dy/dy mice when compared with controls. The intestine, however, exhibited shorter SM cells, similar to what was seen in the lung (Figure 10 A). Unlike the trachea and bronchi, the SM cells in the intestinal wall were arranged in more layers (hyperplasia) resulting in a thicker muscularis wall. In the thinnest areas, the muscular circular layer was 2 ± 1-SM cellthick in dy/dy mouse intestine and 1 ± 1-SM cellthick in control animals. In the thickest sites, the circular layer was 15 ± 3-cell-thick in dydy intestine and 12 ± 2-cell-thick in controls. Reflecting this hyperplasia, immunoblotting showed a slight increase in SM-specific proteins in the intestine of dy/dy mice compared with controls (Figure 10 B).
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Discussion |
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Reciprocal Interactions between LM 2 Chain and Embryonic Mesenchymal Cell Shape
During development, embryonic cells undergo significant changes in shape. In the early stages of embryogenesis, essentially all cells are round, but along with differentiation part of them become spindly whereas others adopt a columnar, cuboidal, or flat configuration. Our studies (1 chain synthesis is induced by epithelial-mesenchymal contact. LM
1 is deposited at the airway epithelial-mesenchymal interface, where it stimulates peribronchial mesenchymal cells to spread and to synthesize SM-specific proteins (
Here we show that this change in cell shape from round to elongated results in the concomitant induction of LM 2 chain expression. In these studies we used different culture approaches to modulate cell shape and to determine its impact on LM
1,
2, ß1, and
1 chain expression. To confirm our in vitro findings, we analyzed tissue fragments microdissected from developing lungs containing either round cells or mostly elongated cells. Our studies demonstrated that, while LM ß1 and
1 chains are equally synthesized by round and elongated cells, LM
2 expression is under the control of cell shape in such a manner that round cells do not synthesize LM
2 chain, either in vivo or in vitro, but its expression is turned on by cell spreading/elongation. Furthermore, the induction of LM
2 chain is a reversible process that can be switched on and off in culture by cyclic changes in cell shape.
It has been shown that limiting the degree of cell spreading in culture may decrease protein secretion (2 chain expression was not suppressed due to nonspecific inhibition in protein synthesis, as indicated by the fact that round and elongated cells synthesized the same amounts of LM ß1/
1 chains. Furthermore, we recently showed that round and elongated cells cultured on microsurfaces have similar metabolic rates, and round cells even synthesize higher levels of
-fetoprotein than their spread/elongated counterparts (
2 chain was not found in undifferentiated mesenchyme freshly isolated from the lungs, confirming its absence in round cells in vivo.
As previously stressed, among the several LM chains analyzed, the effect of cell shape on message and protein levels was specific to LM 2. Message RNA for LM
1,
4,
5, ß1, and
1 chains was not significantly altered by cyclic changes in cell shape, neither were LM ß1 and
1 polypeptide chain levels. In regard to LM
1 chain, its synthesis requires epithelial-mesenchymal cell contact (
1 chain synthesis does not cycle with changes in cell shape.
Functional studies in which embryonic mesenchymal cell spreading/elongation was blocked by a monoclonal antibody against LM 2 chain demonstrated that, once secreted, LM
2 chain becomes a powerful stimulus for cell spreading. The antibody used in our functional studies reacts with the NH2-terminal of LM
2 chain, a region that has been shown to be involved in heparin binding, LM-2 polymerization (
1ß1 and
2ß1 (Collognato and Yurchenco, 1997). The functional role of the NH2 terminus is also underscored by an animal model of congenital muscular dystrophy, the dy2J/dy2J mouse. This mutant animal has a truncated LM
2 chain which lacks only 57 amino acids at its NH2-terminal domain (
1ß1 and
2ß1 are expressed by developing bronchial SM (
2 chainmediated mesenchymal cell spreading. Supporting such a possibility, deletion of integrin
1 by homologous recombination permits normal murine development but gives rise to specific deficit in mesenchymal cell adhesion and spreading (
Role of LM 2 Chain in Bronchial Myogenesis
We recently observed that most embryonic mesenchymal cells are potential SM precursors and if allowed to spread/elongate, they will differentiate into SM cells (2 chain deposition in the extracellular matrix is per se a stimulus for cell spreading. This led us to determine the effect of inhibiting LM
2 chainmediated cell spreading on SM differentiation. Mesenchymal cells exposed to the anti-LM
2 chain antibodies showed a decrease in synthesis of SM-specific proteins when compared with controls. Therefore, a feedback interaction seems to exist between the cells' shape, the levels of LM
2 chain and SM myogenesis.
The molecular pathways linking LM 2 chain, cell shape, and SM myogenesis are currently unknown. One possibility is that by promoting cell spreading/elongation, LM
2 chain stimulates specific cell receptors known to function as mechanotransductors, such as integrins (
2 chaininduced SM myogenesis.
Visceral SM Abnormalities in dy/dy Mice
Dy/dy mice express lower levels of LM 2 chain, due to a spontaneous genetic mutation (
On histological examination of lung sections, bronchial SM cells in dy/dy mice were shorter than in control animals and their lungs contained less SM-specific proteins. The morphological abnormalities diminished in severity from trachea, where the muscle was also underdeveloped, to the distal bronchi. Although the SM changes in the intraparenchymal bronchi were subtle, our current studies seem to indicate that these are sufficient to affect the electrical and contractile properties of dy/dy SM cells in culture (Yang, Y., and L. Schuger, ongoing studies). The SM cells of stomach and intestine were also examined. While no abnormalities were detected in the former, the intestine exhibited shorter SM cells, as seen in the bronchi. However, unlike the airways, the intestinal wall showed more SM cell layers, indicative of compensatory hyperplasia. The latter resulted in a net increase of SM-specific proteins in immunoblots.
It is unclear why some SMs were hyperplastic and others not. One reason could be related to the mechanical challenge sustained by the muscle. Muscles with peristalsis, such as the intestine, are rhythmically stimulated to contract. Since SM cells are able to proliferate, the dy/dy intestinal muscle may overcome the functional deficit caused by low LM 2 chain with an increment in cell number, thus, becoming hyperplastic. In contrast, muscles with minimal mechanical activity, such as the trachea and main bronchi, experience no mechanical challenge and therefore have no need to compensate. Between these two extremes, SMs with moderate contractile activity, such as the intraparenchymal bronchi, may require only intermediate grades of compensation. The gastric and vascular musculature may also fall under this category.
The Reciprocal Interaction between LM 2 Chain and Cell Shape May Play a Role in the Differentiation of Other Tissues
The effect of cell shape on LM 2 chain expression was not restricted to lung mesenchymal cells, but it was seen in other cells as well. Therefore, our findings suggested that cell shapecontrolled LM
2 chain expression may play a broader role in development. In this regard,
2 chain is deposited in the distal airway basement membranes, where epithelial cells are undergoing a change in shape from round/cuboidal to flat/spread (
2 chain is synthesized by elongating mesenchymal cells, but not by epithelial cells that are round/cuboidal in shape. Although neither of these papers intended to draw correlations between the cell shape and LM
2 chain expression, both support the possibility that epithelial cell shape and LM
2 chain may be functionally connected in vivo.
Proposed Roles for LM 1 and
2 in Bronchial Myogenesis
Figure 11 depicts the proposed roles of LM 1 and
2 chains in bronchial myogenesis. Our studies suggest that the development of new epithelial-mesenchymal contacts during lung organogenesis results in the induction of LM
1 chain expression and deposition of LM-1 at the epithelial/mesenchymal interface (
1 (
2 chain and concomitantly to differentiate into SM. Once secreted as part of LM-2, LM
2 chain stimulates spreading and SM differentiation of nearby mesenchymal cells, eventually leading to the establishment of a multilayered visceral muscle. The process of myogenesis could stop when LM
2 chain expression drops dramatically at the end of the pseudo glandular period (
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Footnotes |
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1 Abbreviations used in this paper: LM, laminin; RPA, ribonuclease protection assay; SM, smooth muscle.
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Acknowledgements |
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This work has been supported by NHLBI grant HL-48730 (L. Schuger) and George M. O'Brien Research Center Grant P50 DK45181 (J.M. Miner).
Submitted: 9 August 1999
Revised: 4 November 1999
Accepted: 8 November 1999
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References |
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