Activation of Human Keratinocyte Fibronectin Receptor Function in Relation to Other Ligand-Receptor Interactions

Zhou

European J Oral Sciences

et al. 1992

Immunolocalization of p/ integrins in human gingival epithelium and cultured keratinocytes. Scand J Dent Res 1992; 100: 266-73. Beta 1 integrins are cell surface receptors for extracellular matrix binding. We have recently shown that these receptors may also play a role in cell-cell binding of human epidermal keratinocytes. In this study we used immunofluorescence and confocal laser scanning microscopy to localize beta 1 integrins in frozen sections of human gingiva and cultures of human gingival keratinocytes. The results show that beta 1 integrin polypeptides, localized by monoclonal and polyclonal antibodies, were detected mainly in the basal layer of the keratinized epithelium. There was also scattered staining in connective tissue fibroblasts, nerves, and blood vessel walls. In the basal layer, the integrins were found around the entire periphery of the basal keratinocytes. Furthermore, confocal laser scanning microscopy (CLSM) revealed that most of the staining was in fact localized in dot-like structures at the lateral cell membranes of neighboring basal cells. In cultured human gingival keratinocytes maintained in low calcium (0.15 mM) conditions pi integrins were localized in several different structures: trails which were left behind when the cells moved in culture, dots underneath the cells, around the nucleus, and in cell-cell contacts. The trails were also found to contain fibronectin and type IV collagen but not laminin. Switching the keratinocytes to high calcium (1.2 mM) conditions induced the formation of cell-cell contacts which were strongly positive for pi integrins. No fibronectin or type IV collagen was found in cell-cell contact sites. The results indicate that pi integrins are localized to cell-cell junctions of human gingival keratinocytes both in vivo and in vitro. Cultured cells also express these receptors in cell-matrix contacts indicating a dual role for pi integrins in cell-matrix and cell-cell contacts of human gingival keratinocytes.

Section: Discussionmentioning

confidence: 99%

Immunolocalization of β1 integrins in human gingival epithelium and cultured keratinocytes

Zhou

European J Oral Sciences

et al. 1992

“…The increased synthesis of TSP by proliferating keratinocytes may provide (along with platelets) the matrix that is necessary for subsequent epithelialization. Perhaps TSP should be added to the list of important molecules, such as fibronectin and collagen, which mediate cell-matrix interactions in cutaneous wound healing (30)(31)(32)(33).…”

Section: Discussionmentioning

confidence: 99%

Thrombospondin-induced adhesion of human keratinocytes.

Varani¹,

Nickoloff²,

Riser³

et al. 1988

J. Clin. Invest.

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.

“…Normal human epidermal cell suspensions, which are about 95% keratinocytes, were prepared from human skin biopsies (0.5 × 0.5 cm pieces of full-thickness skin), as described elsewhere [42,43]. Biopsies isolated from plastic surgery procedures (either from thorax, abdomen, or breast) from l2 unrelated consenting adult (aged 15-58 years) male donors were used in this study.…”

Section: Keratinocyte Cell Culturementioning

confidence: 99%

Isolation and Clonal Analysis of Human Epidermal Keratinocyte Stem Cells in Long‐Term Culture

et al. 2003

We developed a procedure for growing normal epidermal keratinocyte stem cells isolated from a single punch biopsy of adult human skin in long-term culture. Primary skin epithelial cells were maintained in collagen-coated plates with irradiated human neonatal foreskin fibroblasts (line HPI.1) as a feeder for more than 120 days, approximately 115 population doublings, without signs of replicative senescence. Clonal analysis revealed the presence of holoclones, meroclones, and paraclones. Only emerging colonies with high proliferative potentials and extensive capacities for division (holoclones and meroclones) were subcultured, favoring the expansion of stem cells and progenitors capable of prolonged self-maintenance when subcloned, thus accounting for the prevailing long-term proliferation of the original culture. We found that meroclones included bipotent progenitors capable of generating both keratinocytes and mucin-producing cells. The numbers of these cells were greater after confluence, suggesting that commitment for their differentiation occurred late in the life of a single clone. On a three-dimensional gelatin matrix and on a collagen layer containing the fibroblast feeder, cells isolated from the expansion of holoclones and meroclones formed stratified cohesive layers of keratinocytes that were able to further differentiate, as in normal skin. These results indicate that our procedure will serve as a valuable tool to study expansion of epidermal stem cells as well as the growth mechanisms and cell products associated with their growth and differentiation.