A preformed basal lamina alters the metabolism and distribution of hyaluronan in epidermal keratinocyte "organotypic" cultures grown on collagen matrices

Abstract

A rat epidermal keratinocyte (REK) line which exhibits histodifferentiation nearly identical to the native epidermis when cultured at an air-liquid interface was used to study the metabolism of hyaluronan, the major intercellular macromolecule present in basal and spinous cell layers. Two different support matrices were used: reconstituted collagen fibrils with and without a covering basal lamina previously deposited by canine kidney cells. REKs formed a stratified squamous, keratinized epithelium on both support matrices. Hyaluronan and its receptor, CD44, colocalized in the basal and spinous layers similar to their distribution in the native epidermis. Most (approximately 75%) of the hyaluronan was retained in the epithelium when a basal lamina was present while most (approximately 80%) diffused out of the epithelium in its absence. While REKs on the two matrices synthesized hyaluronan at essentially the same rate, catabolism of this macromolecule was much higher in the epithelium on the basal lamina (half-life approximately 1 day, similar to its half-life in native human epidermis). The formation of a true epidermal compartment in culture bounded by the cornified layer on the surface and the basal lamina subjacent to the basal cells provides a good model within which to study epidermal metabolism.

Low Dose Ultraviolet B Irradiation Increases Hyaluronan Synthesis in Epidermal Keratinocytes via Sequential Induction of Hyaluronan Synthases Has1–3 Mediated by p38 and Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Signaling*

“…Metabolic Labeling-In the organotypic cultures, hyaluronan synthesis was quantified using metabolic labeling as described previously (51). The organotypic cultures were irradiated as described above and incubated for 24 h in 1 ml of medium containing 1% FBS and 20 Ci/ml [ 3 H]glucosamine and 100 Ci/ml [ 35 S]Na 2 SO 4 (both from PerkinElmer Life Sciences).…”

“…The samples were subsequently purified and quantified for hyaluronan and chondroitin sulfate as described previously (42,51). The samples were digested overnight at 60°C with papain (Sigma-Aldrich), boiled, and centrifuged.…”

“…Molecular Mass Distribution of Hyaluronan-To analyze the size distribution of hyaluronan secreted in the medium, gel filtration on a 1 ϫ 30-cm Sephacryl S-1000 column (HR 10/30; GE Healthcare) was done as described previously (51). 150 mM sodium acetate buffer (pH 6.8) containing 0.1% CHAPS, at a flow rate of 24 ml/h, was used for elution.…”

“…However, the metabolism of epidermal hyaluronan is more efficient in organotypic cultures (51,75) than in monolayers. 4 Keratinocytes in the normal epidermis produce hyaluronan into the narrow intercellular space, where it is probably more accessible to the degradative machinery (receptors, enzymes, and ROS), compared with monolayers, where hyaluronan is mostly released into a large volume of culture medium.…”

The DP-1 Transcription Factor Is Required for Keratinocyte Growth and Epidermal Stratification

“…REK Organotypic Cultures-REK cells were plated on a basal lamina-containing collagen substrate and cultured to allow stratification as described previously (36). In brief, to first generate a basal lamina, confluent MDCK monolayers were cultured onto collagen gels in a Transwell insert for 22 days.…”