Limits...
Endogenous laminin is required for human airway smooth muscle cell maturation.

Tran T, McNeill KD, Gerthoffer WT, Unruh H, Halayko AJ - Respir. Res. (2006)

Bottom Line: While the effects of extracellular matrix (ECM) components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear.Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP) significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation.Thus, human ASM maturation appears to involve regulated endogenous expression of a select set of laminin chains that are essential for accumulation of contractile phenotype myocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, University of Manitoba, Winnipeg, MB, Canada. ttran@mich.ca

ABSTRACT

Background: Airway smooth muscle (ASM) contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM) components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells.

Methods: Human ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured.

Results: Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP) significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of alpha2, beta1 and gamma1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype.

Conclusion: While earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the first time that endogenously expressed laminin is required for ASM maturation to the contractile phenotype. As endogenously expressed laminin chains alpha2, beta1 and gamma1 are uniquely increased during myocyte maturation, these laminin chains may be key in this process. Thus, human ASM maturation appears to involve regulated endogenous expression of a select set of laminin chains that are essential for accumulation of contractile phenotype myocytes.

Show MeSH

Related in: MedlinePlus

Effect of laminin-competing peptides (YIGSR, 1 μM and GRGDSP, 1 μM) on (A) desmin and (B) calponin protein abundance following 7-day serum deprivation. YIGSR = peptide derived from the amino acid sequence of the β1 chain of the major receptor binding site in laminin; GRGDSP = amino acid sequence within fibronectin and other extracellular proteins that mediates cell attachment; GRADSP = inactive peptide for GRGDSP. Grouped data represent results obtained from three different cultures. * P < 0.05, compared with Day 0; † P < 0.05 compared with Day 7 response in the absence of peptide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1586013&req=5

Figure 1: Effect of laminin-competing peptides (YIGSR, 1 μM and GRGDSP, 1 μM) on (A) desmin and (B) calponin protein abundance following 7-day serum deprivation. YIGSR = peptide derived from the amino acid sequence of the β1 chain of the major receptor binding site in laminin; GRGDSP = amino acid sequence within fibronectin and other extracellular proteins that mediates cell attachment; GRADSP = inactive peptide for GRGDSP. Grouped data represent results obtained from three different cultures. * P < 0.05, compared with Day 0; † P < 0.05 compared with Day 7 response in the absence of peptide.

Mentions: We first assessed phenotype maturation by measuring the abundance of stringent contractile phenotype marker proteins desmin and calponin [5,8]. Under basal conditions, 16 hrs after replating myocytes from confluent cultures in DMEM containing 0.5% FBS, HASM cells expressed low levels of desmin and calponin. However, following 7-days in serum deficient conditions, both desmin and calponin protein increased markedly, exhibiting a doubling in abundance (P < 0.05, Figure 1). This expression pattern is consistent with phenotype maturation of ASM cell myocytes that we have described previously for both hTERT immortalized cells and primary cultured airway smooth muscle cells [5,8,25].


Endogenous laminin is required for human airway smooth muscle cell maturation.

Tran T, McNeill KD, Gerthoffer WT, Unruh H, Halayko AJ - Respir. Res. (2006)

Effect of laminin-competing peptides (YIGSR, 1 μM and GRGDSP, 1 μM) on (A) desmin and (B) calponin protein abundance following 7-day serum deprivation. YIGSR = peptide derived from the amino acid sequence of the β1 chain of the major receptor binding site in laminin; GRGDSP = amino acid sequence within fibronectin and other extracellular proteins that mediates cell attachment; GRADSP = inactive peptide for GRGDSP. Grouped data represent results obtained from three different cultures. * P < 0.05, compared with Day 0; † P < 0.05 compared with Day 7 response in the absence of peptide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1586013&req=5

Figure 1: Effect of laminin-competing peptides (YIGSR, 1 μM and GRGDSP, 1 μM) on (A) desmin and (B) calponin protein abundance following 7-day serum deprivation. YIGSR = peptide derived from the amino acid sequence of the β1 chain of the major receptor binding site in laminin; GRGDSP = amino acid sequence within fibronectin and other extracellular proteins that mediates cell attachment; GRADSP = inactive peptide for GRGDSP. Grouped data represent results obtained from three different cultures. * P < 0.05, compared with Day 0; † P < 0.05 compared with Day 7 response in the absence of peptide.
Mentions: We first assessed phenotype maturation by measuring the abundance of stringent contractile phenotype marker proteins desmin and calponin [5,8]. Under basal conditions, 16 hrs after replating myocytes from confluent cultures in DMEM containing 0.5% FBS, HASM cells expressed low levels of desmin and calponin. However, following 7-days in serum deficient conditions, both desmin and calponin protein increased markedly, exhibiting a doubling in abundance (P < 0.05, Figure 1). This expression pattern is consistent with phenotype maturation of ASM cell myocytes that we have described previously for both hTERT immortalized cells and primary cultured airway smooth muscle cells [5,8,25].

Bottom Line: While the effects of extracellular matrix (ECM) components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear.Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP) significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation.Thus, human ASM maturation appears to involve regulated endogenous expression of a select set of laminin chains that are essential for accumulation of contractile phenotype myocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, University of Manitoba, Winnipeg, MB, Canada. ttran@mich.ca

ABSTRACT

Background: Airway smooth muscle (ASM) contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM) components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells.

Methods: Human ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured.

Results: Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP) significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of alpha2, beta1 and gamma1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype.

Conclusion: While earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the first time that endogenously expressed laminin is required for ASM maturation to the contractile phenotype. As endogenously expressed laminin chains alpha2, beta1 and gamma1 are uniquely increased during myocyte maturation, these laminin chains may be key in this process. Thus, human ASM maturation appears to involve regulated endogenous expression of a select set of laminin chains that are essential for accumulation of contractile phenotype myocytes.

Show MeSH
Related in: MedlinePlus