Brightfield proximity ligation assay reveals both canonical and mixed transforming growth factor-ß/bone morphogenetic protein Smad signaling complexes in tissue sections.

Transforming growth factor-ß (TGF-ß) is an important regulator of cellular homeostasis and disease pathogenesis. Canonical TGF-ß signaling occurs through Smad2/3-Smad4 complexes; however, recent in vitro studies suggest that elevated levels of TGF-ß may activate a novel mixed Smad complex (Smad2/3-Smad1/5/9), which is required for some of the pro-oncogenic activities of TGF-ß. To determine if mixed Smad complexes are evident in vivo, we developed antibodies that can be used with a proximity ligation assay to detect either canonical or mixed Smad complexes in formalin-fixed paraffin-embedded sections. We demonstrate high expression of mixed Smad complexes in the tissues from mice genetically engineered to express high levels of TGF-ß1. Mixed Smad complexes were also prominent in 15-16 day gestation mouse embryos and in breast cancer xenografts, suggesting important roles in embryonic development and tumorigenesis. In contrast, mixed Smad complexes were expressed at extremely low levels in normal adult mouse tissue, where canonical complexes were correspondingly higher. We show that this methodology can be used in archival patient samples and tissue microarrays, and we have developed an algorithm to quantitate the brightfield read-out. These methods will allow quantitative analysis of cell type-specific Smad signaling pathways in physiological and pathological processes.

Spontaneous skin erosions and reduced skin and corneal wound healing characterize CLIC4(NULL) mice.

Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-ß (TGF-ß) signaling, and mice lacking components of the TGF-ß signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-ß pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of ß4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-ß was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-ß. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-ß pathway.