ABSTRACT
We have previously demonstrated that the EP1 subtype of PGE2 receptor is expressed in the differentiated compartment of normal human epidermis and is coupled to intracellular calcium mobilization. We therefore hypothesized that the EP1 receptor is coupled to keratinocyte differentiation. In in vitro studies, radioligand binding, RT-PCR, immunoblot and receptor agonist-induced second messenger studies demonstrate that the EP1 receptor is up-regulated by high cell density in human keratinocytes and this up-regulation precedes corneocyte formation. Moreover, two different EP1 receptor antagonists, SC51322 and AH6809, both inhibited corneocyte formation. SC51322 also inhibited the induction of differentiation-specific proteins, cytokeratin K10 and epidermal transglutaminase. We next examined the immunolocalization of the EP1 receptor in non-melanoma skin cancer in humans. Well-differentiated SCCs exhibited significantly greater membrane staining, while spindle cell carcinomas and BCCs had significantly decreased membrane staining compared with normal epidermis. This data supports a role for the EP1 receptor in regulating keratinocyte differentiation.
Subject(s)
Cell Differentiation , Keratinocytes/cytology , Keratinocytes/metabolism , Receptors, Prostaglandin E/classification , Receptors, Prostaglandin E/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Animals , Calcium/metabolism , Humans , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Receptors, Prostaglandin E/antagonists & inhibitors , Receptors, Prostaglandin E/biosynthesis , Receptors, Prostaglandin E, EP1 Subtype , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tumor Cells, Cultured , Xanthones/pharmacologyABSTRACT
Human involucrin (hINV) is a constituent of the scaffolding of the cornified envelope. In the present study, we describe an in vitro model system to study the role of hINV in scaffold formation. We characterize the in vitro cross-linking of full-length (585 amino acid) recombinant hINV, rhINV(1-585). When reacted with detergent-solubilized, particulate transglutaminase type 1 (TG1) or partially purified type 2 transglutaminase (TG2), rhINV(1-585) functions as a TG substrate in a calcium-dependent manner. When the reaction is supplemented with 14C-putrescine tracer, the radiolabeled cosubstrate is incorporated into a high-molecular-weight product in a calcium-, rhINV(1-585)- and time-dependent manner. 35S-rhINV(1-585) is also cross-linked to form a high-molecular-weight product. These results suggest that rhINV(1-585) is extensively multimerized. Products having a molecular weight smaller than authentic rhINV(1-585) are also formed, providing evidence for intramolecular cross-link formation. Transmission electron microscopy of cross-linked product reveals immunoreactive large-molecular-weight loop-string-loop and branched structures. Our studies (1) show that rhINV(1-585) is a substrate for both TG1 and TG2, (2) indicate that rhINV(1-585) can be cross-linked to form macromolecular products having distinct structural features, (3) demonstrate that rhINV(1-585) forms intramolecular cross-links when hINV concentration is limiting and (4) establish that hINV possesses reactive Gln and Lys residues.
Subject(s)
Cross-Linking Reagents/chemistry , Protein Precursors/chemistry , Bacteria/metabolism , Cells, Cultured , Epidermis/metabolism , Genetic Vectors , Humans , Immunochemistry , Keratinocytes/drug effects , Keratinocytes/ultrastructure , Microscopy, Electron , Molecular Weight , Protein Precursors/genetics , Protein Precursors/isolation & purification , Putrescine/metabolism , Recombinant Proteins/chemistry , Sulfur Radioisotopes , Transglutaminases/metabolismABSTRACT
Involucrin (hINV) is an important structural component of the keratinocyte cornified envelope that is expressed early in the keratinocyte differentiation process and is thought to be a component of the initial envelope scaffolding. We have previously shown that cyanogen bromide (CNBr) cleavage of cornified envelopes isolated from cultured foreskin keratinocytes releases several discrete involucrin-immunoreactive peptides. In this study, we compare the pattern of release of immunoreactive hINV fragments from envelopes prepared from human breast skin and foreskin, and from spontaneous and induced envelopes prepared from cultured keratinocytes. We also identify one of the released products. Envelopes prepared from human breast skin or foreskin, or spontaneous or induced envelopes prepared from cultured cells differ significantly in structure. The envelopes isolated from epidermis appear to be structurally complete, whereas spontaneous envelopes appear less complete and the induced envelopes appear to be the least complete. In spite of these structural differences, CNBr cleavage releases an identical quartet of hINV-immunoreactive peptides migrating between 68 and 81 kDa from each preparation. Immunoblots indicate that the quantity of hINV-immunoreactive material released per microg of envelope protein is as follows: induced > spontaneous > foreskin > breast skin. The fastest migrating peptide (68 kDa) comigrates with a peptide that is released after CNBr cleavage of bacterially produced-recombinant hINV. Amino-terminal amino acid sequencing of this peptide from recombinant hINV and from the cornified envelopes yields the sequence G-Q-L-K-H-L-E-Q-Q-E-G-Q-P-K-H. These results suggest that this fragment is the 275-amino acid segment of hINV beginning at G311 and extending to K585, and that this peptide is not crosslinked to another protein. These results indicate that a population of the envelope-associated hINV present in cultured and in vivo keratinocytes is crosslinked in the amino-terminal half. It is possible that this species represents an early intermediate in the involucrin crosslinking process.
