Reduced Migration, Altered Matrix and Enhanced TGFβ1 Signaling are Signatures of Mouse Keratinocytes Lacking Sdc1

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

Suspension-Induced Murine Keratinocyte Differentiation Is Mediated by Calcium

Modulating extracellular Ca2+ (Cao) and suspension culture are two frequently used methods to induce maturation of cultured human and mouse keratinocytes. To determine if the two methods share a common mechanism, changes in Ca2+ metabolism were studied in suspension cultures of mouse keratinocytes. Spontaneously detached and suspensioncultured keratinocytes in 0.05mM Ca2+ medium express markers of suprabasal differentiation, while 0.05mM Ca2+ is not permissive for marker expression by attached keratinocytes. Intracellular free Ca2+ (Cai increased rapidly after placing keratinocytes in suspension in 0.05mM Ca2+, reaching levels up to 3to 4-fold higher than Cai in attached cells after 4-5h. In suspended cells, the increase in Cai was associated with a 2- to 6-fold increase in Ca2+ transport across plasma membrane as well as depletion of intracellular Ca2+-stores. Differentiation marker expression and terminal differentiation were inhibited in suspension-cultured keratinocytes by preventing the rise of Cai using either 1,2-bis(o-aminophenoxy)-ethaneN,N,N',N'-tetraacetic acid to chelate intracellular Ca2+ or ethyleneglycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid to reduce Cao. Together, these results indicate that a rise in Cai is a common mechanism controlling differentiation in cultured mouse keratinocytes, and suspension of keratinocytes enhances Ca2+ transport and alters intracellular Ca2+ sequestration producing a rise in Cai