HoxB-5 down regulation alters Tenascin-C, FGF10 AND HoxB gene expression patterns in pseudoglandular period fetal mouse lung

Abstract

Organ-specific patterning is partly determined by Hox gene regulatory interactions with the extracellular matrix (ECM), cell adhesion and fibroblast growth factor (FGFs) signaling pathways but coordination of these mechanisms in lung development is unknown. We have previously shown that Hoxb-5 affects airway patterning during lung morphogenesis. Hoxb-5 regulation in fetal lung affects ECM expression of tenascin-C and alters FGF10 spatial and cellular expression. To test this hypothesis, gestational day 13.5 (Gd13.5) fetal mouse lung fibroblasts and whole lungs were cultured with Hoxb-5-specific small interfering RNA (siRNA). Western blots showed that siRNA-down regulation of Hoxb-5 led to decreased tenascin-C and FGF10 and was associated with increased Hoxb-4 and decreased Hoxb-6 protein levels. Hoxa-5 protein levels were not affected. Hoxb-5 siRNA-treated whole lung cultures had a significant decrease in total lung and peripheral branching region surface area. Immunostaining showed negligible levels of Hoxb-5 protein and tenascin-C, and loss of FGF10 spatial restriction. We conclude that Hoxb-5 helps regulate lung airway development through modulation of ECM expression of tenascin-C. ECM changes induced by Hoxb-5 may affect mesenchymal-epithelial cell signaling to alter spatial and cellular restriction of FGF10. Hoxb-5 may also affect lung airway branching indirectly by cross regulation of other Hoxb genes.

Aberrant cell adhesion molecule expression in human bronchopulmonary sequestration and congenital cystic adenomatoid malformation

“…Mouse lung fibroblasts cultures. To compliment the studies in human tissue, we evaluated ␣ 2-integrin expression in E13.5 (E0.5 identification of vaginal plug, term is 19 days) mouse lung fibroblast cultures in which we have previously reported Hoxb5 downregulation by Hoxb5-specific siRNA molecules (60). The animal protocols were approved by Tufts University School of Medicine IACUC.…”

“…Fetal mouse lungs were isolated, and fetal mouse lung fibroblasts were cultured with no treatment, TKO transfection vehicle (Mirus, Madison, WI), scrambled siRNA control, or Hoxb5-specific siRNA. This siRNA protocol and the sequences for Hoxb5 siRNA and scrambled siRNA control have been published (60). After 72 h of treatment, mouse fetal lung fibroblast cultures were harvested and prepared for Western blot analysis as described below.…”

“…Immunostaining reagents were from Vector (Burlingame, CA). siRNA molecules were purchased from Ambion as previously described (60). All other reagents were purchased from Fisher (Pittsburgh, PA) unless otherwise specified.…”

“…This antibody that reacts against the extracellular domain of ␣ 2-integrin does not crossreact with mouse tissue. ␣ 2-integrin protein levels in mouse fetal lung fibroblasts with Hoxb5-specific siRNA downregulation of Hoxb5 protein were compared with ␣2-integrin levels in scramble siRNAtreated cells as previously described (60).…”

“…This suggests that Hox genes that help determine embryonic and organ-specific patterning evolved along with integrins that help recognize cellcell and cell-matrix interactions coordinating cell positioning and cellular communication in developing and mature organs and tissues (13,28,63). In mice, we have reported that regulated spatial and temporal expression of the Hox protein, Hoxb5, helps regulate airway branching patterns partially through regulation of cell matrix molecules (57,60,61). We have also shown that HoxB5 (nomenclature for human Hoxb5) is expressed in human lung development in a similar pattern to that seen in mouse and that human BPS and CCAM tissue has abnormally high levels of HoxB5 protein, analogous to the canalicular stage of lung development and significantly increased over age and lung development stage-matched controls (59,64).…”