In wounded barrier epithelia, front row cells often undergo partial or complete dissolution of cell-cell contacts and acquire some mesenchymal characteristics. This functions to disaggregate epithelial units and reshape epithelia for movement (17
). Epithelia in transition lose polarity, intercellular junctions, and down-regulate cytokeratin filaments in order to rearrange F-actin stress fibers and express filopodia and lamellopodia (17
). Several lines of evidence show that SPRR2 expression is associated with this process.
Forced non-coordinate expression of SPRR2A in SG231 cells, in vitro, can lead to a front row phenotype. Since SG231 is a cell line the results should be interpreted with some caution. But non-neoplastic BEC with similar phenotypic characteristics were also observed in low-confluency primary mouse BEC cultures and in atypical ductules in PBC. SPRR2A is also expressed exclusively in the remodeling biliary tree after bile duct ligation (5
), and in many injured and remodeling barrier epithelia from the lung, skin, uterus, and intestine after exposure to injury and stress (). It seems reasonable, therefore, to suggest that SPRR2A upregulation is a stress-related response that helps to protect barrier epithelium from oxidative damage and prepares it for remodeling. It would have been ideal to conditionally block biliary SPRR expression to further investigate SPRR function. But this approach has not been feasible because of redundancy and regulatory diversity in the SPRR gene locus.
Molecular studies show that non-coordinate BEC expression makes SPRR2A available for binding to SH3 domain-containing proteins (25
), such as c-Src, c-Yes, and abl. Other binding partners exist. These particular molecules, however, contribute significantly to formation and maintenance of intercellular junctions, shape changes (20
), and to acquisition of some mesenchymal characteristics (19
As seen in the protein binding array, promiscuity of SH3 ligand binding enables numerous potential interactions and facilitates assembly of large multi-molecular complexes. This can lead to context specific effects (28
). It is likely, therefore, that SPRR expression might not always produce the same effects, but be dependent on the cell proteome at the time of SPRR expression. This likely accounts for some of the variability seen in our different SPRR2A clones and that Sprr2 is expressed in damaged septal bile ducts and in atypical cholangioles, which differ in their vimentin expression.
Spindle-shaped atypical ductules in PBC can acquire some mesenchymal characteristics similar to the SPRR2A transfectants and low confluency mouse BEC. In embryonic liver ductal plate BEC transiently express vimentin (29
) and down-regulate membranous E-cadherin (30
) when they “invade” the portal connective tissue to form mature intrahepatic bile ducts. SPRR2A is also highly upregulated when mouse hepatoblasts transform into BEC after exposure to Matrigel (http://www.ncbi.nlm.nih.gov/projects/geo/gds/gds_browse.cgi?gds=970
). During injury and repair (1
), BEC can transform from polarized cuboidal epithelial cells into spindle-shaped migratory cells that can express vimentin (31
). However, since BEC appear to retain some epithelial characteristics during this process, BEC EMT may not be exactly the same as described for kidney tubular epithelial cells (17
). Instead, acquisition of mesenchymal characteristics in BEC may be “meta-stable” because they appear to revert to an epithelial phenotype when migration/repair is complete. More study is needed on this topic.
Among liver diseases tested, SPRR2A is most highly expressed in BEC of small atypical ductules from PBC livers, and to a lesser extent in other diseases. It was clear, however, that EMT changes were more common in atypical ductular reactions. It is likely, therefore, that the heterogeneity of BEC in large versus small ducts (32
) accounts for this difference. In the uterus, however, SPRR2A is highly upregulated in an estrogen-dependent fashion at the blastocyst implantation site where decreased intercellular junctions, desmoplakin, and cytokeratin are also seen (33
). The possibility that estrogen and IL-6 co-dependent BEC barrier modifications might be involved in PBC pathogenesis is also worthy of further study.
This study focuses specifically on BEC but the ubiquity and magnitude of non-coordinate SPRR expression in non-squamous tissues, suggests that it is part of a generic stress/remodeling response. Although not described in mammalian cells, proline utilization is widely recognized as an anti-stress response in plants (34
). Proline residues in proline-rich proteins can be converted to either 4- or 5-hydroxy proline by ROS and these changes will not cause cleavage of the polypeptide or damage the amino acid backbone (34
In conclusion, our studies suggest that SPRR upregulation is a widely utilized stress and remodeling response which contributes to barrier function, wound repair and increase resistance to injury. This may explain why the gene locus is so rapidly diverging (9
) and a “hot-spot” for non-synonymous substitutions (8