Expression of β ig-h3 by Human Bronchial Smooth Muscle Cells

Abstract

Bronchial smooth muscle cells play a central role in normal lung physiology by controlling airway tone. In addition, airway smooth muscle hyperplasia and hypertrophy are important factors in the pathophysiology of asthma. In this study, expression of betaig-h3, a recently identified component of the extracellular matrix (ECM), was investigated in primary human bronchial smooth muscle (HBSM) cells. Northern blot analysis demonstrated that treatment of cultured HBSM cells with transforming growth factor-beta1 resulted in a 4- to 5-fold increase in the steady-state level of betaig-h3 messenger RNA. Western blot analysis of secreted proteins using monospecific antibodies generated against peptide sequences found in the N- and C-terminal regions of the protein identified several isoforms having apparent mass of 70-74 kD. Immunohistochemical analysis of human lung localized betaig-h3 to the vascular and airway ECM, and particularly to the septal tips of alveolar ducts and alveoli, suggesting that it may have a morphogenetic role. Analysis of cultured HBSM cells identified betaig-h3 in both the ECM as well as the cytoplasm, and surprisingly also in the nucleus. These results demonstrate that betaig-h3 is produced by resident lung cells, is a component of lung ECM, and may play an important role in lung structure and function. The presence of this protein in nuclei suggests that it may have regulatory functions in addition to its role as a structural component of lung ECM.

The Transforming Growth Factor-β-inducible Matrix Protein βig-h3 Interacts with Fibronectin

“…Antibody Production and Purification-Anti-␤ig-h3 antibodies 1073 and 1077 were developed in rabbits against synthetic peptides present in the N-and C-terminal regions of human ␤ig-h3 and affinity-purified as described previously (12). For immobilization, anti-␤ig-h3 antibody 1077 and preimmune rabbit IgG were covalently attached to Affi-Gel-10 support resin following the protocols recommended by the manufacturer.…”

“…Western Blot Analysis-Proteins were resolved on Novex 8 -16% polyacrylamide gradient gels under reducing conditions and transferred to nitrocellulose membranes (12,13). After transfer, the membranes were blocked in PBST and 5% nonfat dry milk and subsequently incubated with anti-␤ig-h3 antibodies diluted 1:2000 in PBST, 0.3% bovine serum albumin.…”

“…Analysis of Conditioned Medium-We have previously reported that human lung and bladder cells secrete ␤ig-h3 into culture medium (12,13). An analysis of conditioned medium from these cells by Bio Gel P200 gel filtration chromatography (exclusion limit ϳ200 kDa) revealed that the bulk of ␤ig-h3 elutes with the void volume and an apparent M r Ͼ 200 (data not shown).…”

“…It was also present in capsule and tubule basement membranes of developing kidney and reticular fibers in fetal spleen. We recently reported that ␤ig-h3 is expressed by primary human lung and bladder smooth muscle cells and fibroblast cells in vitro where it is secreted into the culture medium and incorporated in the matrix (12,13). In human tissue, the protein is deposited in the matrix surrounding individual smooth muscle cells in the bladder detrusor (13), in septal tips of alveolar ducts, and in the walls of alveoli and bronchioles in lung (12).…”

“…Although the precise physiological function of ␤ig-h3 is not known, it has been suggested that it interconnects different matrix components and resident cells (11)(12)(13) and consequently serves as a bifunctional linker protein. In this report, we demonstrate that ␤ig-h3 supports cell attachment and spreading, induces actin stress fiber formation, and binds fibronectin, an abundant glycoprotein of the extracellular matrix.…”