Thrombospondin-induced adhesion of human keratinocytes.

Abstract

Human epidermal keratinocytes obtained from normal skin attached and spread on thrombospondin (TSP)-coated plastic dishes but failed to attach and spread on untreated plastic culture dishes or dishes coated with fibronectin or laminin. These cells produced minimal amounts of immunoreactive TSP. Keratinocytes established in culture on MCDB 153 medium and maintained for one to three passages in an undifferentiated state by continued cultivation in this low Ca2+-containing medium attached and spread on plastic dishes as well as on TSP-coated dishes. These cells also secreted significant amounts of TSP into the culture medium. When the keratinocytes were incubated for one day in MCDB 153 medium supplemented with high Ca2+ or in MEM (which also contains high Ca2"), there was decreased secretion of TSP into the culture medium concomitant with a reduction in attachment and spreading on plastic culture dishes.Proteolytic fragments of TSP were examined for stimulation of keratinocyte attachment and spreading. A 140-kd fragment produced by removal of the 25-kd heparin-binding domain had similar activity to the intact molecule while the 25-kd fragment was without effect. Further proteolytic treatment of the 140-kd fragment gave rise to a fragment consisting of 120 kd and 18-D moieties held together in disulphide linkage. This fragment did not support attachment or spreading.This study reveals that normal epidermal keratinocytes grown under conditions that maintain the undifferentiated state are able to produce TSP and utilize it as an attachment factor. When keratinocytes are grown under conditions that promote differentiation, ability to produce and utilize TSP is diminished. Since TSP is present at the dermal-epidermal junction and because TSP promotes keratinocyte attachment and spreading, this molecule may play an important role in maintaining normal growth of the basal cell layer and may also participate in reepithelialization during wound repair.

Antisense-mediated reduction in thrombospondin reverses the malignant phenotype of a human squamous carcinoma.

“…In vitro, undifferentiated, rapidly-proliferating epidermal keratinocytes synthesize large amounts of TSP and incorporate it into the ECM. When the cells are induced to differentiate, TSP production is reduced concomitantly with inhibition ofproliferation (7,14). In addition, stimulation of proliferation in quiescent keratinocytes by addition of epidermal growth factor (EGF) or somatomedin C is associated with a rapid induction of TSP synthesis (1 1).…”

“…Thus TSP may serve to modulate growth ofcells of both mesodermal and ectodermal origin. The mechanism by which this occurs is unknown, although it has been shown that endogenously-synthesized TSP serves as a potent adhesion factor for both normal and malignant squamous epithelial cells (5)(6)(7)(8)(9). By altering the synthesis of this protein, we may have interfered with the capacity of the cells to correctly interact with their matrix and altered their ability to receive and process growth controlling signals.…”

“…Functionally, TSP acts as a growth supportive matrix component. TSP has been shown to facilitate cell attachment and serve as an adhesion factor for many cell types (5)(6)(7)(8)(9). Several converging lines of evidence point to a role for TSP in controlling cellular proliferation.…”

“…Third, TSP is found in areas of cellular proliferation in developing mouse embryos (12). Fourth, TSP is made in significant quantities by proliferating undifferentiated human epidermal keratinocytes (13), and its production falls in cells induced to differentiate by a variety of means including exposure to high external Ca2" concentrations, exposure to interferon-y or depletion of growth supplements from the culture medium (7,14).…”

Intraglomerular pressure and mesangial stretching stimulate extracellular matrix formation in the rat.

“…The amount ofspecific ECM components secreted into the culture medium was quantified by ELISA, using a modification of a procedure previously described (23). Harvested culture medium was clarified by low speed centrifugation and, except for measurement of laminin, diluted 1/2 in 1% FCS-containing fresh medium and 100 ,d…”