Reduced migration, altered matrix and enhanced TGFβ1 signaling are signatures of mouse keratinocytes lacking Sdc1

Abstract

We have reported previously that syndecan-1 (Sdc1)-null mice show delayed re-epithelialization after skin and corneal wounding. Here, we show that primary keratinocytes obtained from Sdc1-null mice and grown for 3-5 days in culture are more proliferative, more adherent and migrate more slowly than wt keratinocytes. However, the migration rates of Sdc1-null keratinocytes can be restored to wild-type levels by replating Sdc1-null keratinocytes onto tissue culture plates coated with fibronectin and collagen I, laminin (LN)-332 or onto the matrices produced by wild-type cells. Migration rates can also be restored by treating Sdc1-null keratinocytes with antibodies that block α6 or αv integrin function, or with TGFβ1. Antagonizing either β1 integrin function using a function-blocking antibody or TGFβ1 using a neutralizing antibody reduced wild-type keratinocyte migration more than Sdc1-null keratinocyte migration. Cultures of Sdc1-null keratinocytes accumulated less collagen than wild-type cultures but their matrices contained the same amount of LN-332. The Sdc1-null keratinocytes expressed similar total amounts of eight different integrin subunits but showed increased surface expression of αvβ6, αvβ8, and α6β4 integrins compared with wild-type keratinocytes. Whereas wild-type keratinocytes increased their surface expression of α2β1, αvβ6, αvβ8, and α6β4 after treatment with TGFβ1, Sdc1-null keratinocytes did not. Additional data from a dual-reporter assay and quantification of phosphorylated Smad2 show that TGFβ1 signaling is constitutively elevated in Sdc1-null keratinocytes. Thus, our results identify TGFβ1 signaling and Sdc1 expression as important factors regulating integrin surface expression, activity and migration in keratinocyte and provide new insight into the functions regulated by Sdc1.

Accelerated re-epithelialization inDpr2-deficient mice is associated with enhanced response to TGFβ signaling

“…Integrin αvβ6 gene expression is increased significantly in wound keratinocytes, and has been reported to promote keratinocyte migration (Huang et al, 1998;Thomas et al, 2001). Consistently with previous studies (Gailit et al, 1994;Stepp et al, 2007;Zambruno et al, 1995), the results of our study show that TGFβ1 upregulated the gene expression of integrins αv, β1 and β6 in both wild-type and Dpr2-deficient keratinocytes. However, integrin expression levels were significantly increased in Dpr2 -/-cells compared with wild-type cells in response to TGFβ1 treatment.…”

“…This inconsistency might be due to the use of different animal models. In K14-Smad2 (Hosokawa et al, 2005) and K14-TGFβ1 (Yang et al, 2001) transgenic mice or in Sdc1-null mice (Stepp et al, 2002;Stepp et al, 2007), skin keratinocytes during the woundhealing process might have TGFβ1-signaling levels above a threshold at which cell migration is inhibited, probably because of alterations in the extracellular matrix.…”

“…Primary mouse keratinocytes were isolated from skin of newborn Dpr2 -/-or control mice, as described (Stepp et al, 2007). Full-thickness skin was treated with 0.25% dispase overnight at 4°C.…”

MMP9 cleavage of the β4 integrin ectodomain leads to recurrent epithelial erosions in mice

“…Primary mouse keratinocytes were derived from neonatal Balb/c mouse pups and were cultured as described previously (Stepp et al, 2007) and used after 3 days of culture in low-calcium medium. The human corneal and limbal epithelial (HCLE) cell line was a kind gift from Ilene Gipson (Schepen's Eye Research Institute, Harvard Medical School, Boston, MA).…”

“…Epidermal keratinocytes were cultured for 3 days in low-calcium medium as described (Stepp et al, 2007). Purified recombinant mouse MMP2, MMP3 or MMP9 (all R&D Systems) were used after a 1 hour activation step with 4-aminophenylmercuric acetate (APMA) (Sigma) as described by the manufacturer.…”