JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells

Am J Physiol Gastrointest Liver Physiol. 2008 Feb;294(2):G576-88. doi: 10.1152/ajpgi.00159.2007. Epub 2007 Dec 20.

Abstract

Junctional adhesion molecule (JAM) is involved in tight junction (TJ) formation in epithelial cells. Three JAMs (A, B, and C) are expressed in rat hepatocytes, but only rat JAM-A is present in polarized WIF-B cells, a rat-human hepatic line. We used knockdown (KD) and overexpression in WIF-B cells to determine the role of JAM-A in the development of hepatic polarity. Expression of rat JAM-A short hairpin RNA resulted in approximately 50% KD of JAM-A and substantial loss of hepatic polarity, as measured by the absence of apical cysts formed by adjacent cells and sealed by TJ belts. When inhibitory RNA-resistant human JAM-A (huWT) was expressed in KD cells, hepatic polarity was restored. In contrast, expression of JAM-A that either lacked its PDZ-binding motif (huDeltaC-term) or harbored a point mutation (T273A) did not complement, indicating that multiple sites within JAM-A's cytoplasmic tail are required for the development of hepatic polarity. Overexpression of huWT in normal WIF-B cells unexpectedly blocked WIF-B maturation to the hepatic phenotype, as did expression of three huJAM-A constructs with single point mutations in putative phosphorylation sites. In contrast, huDeltaC-term was without effect, and the T273A mutant only partially blocked maturation. Our results show that JAM-A is essential for the development of polarity in cultured hepatic cells via its possible phosphorylation and recruitment of relevant PDZ proteins and that hepatic polarity is achieved within a narrow range of JAM-A expression levels. Importantly, formation/maintenance of TJs and the apical domain in hepatic cells are linked, unlike simple epithelia.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Adhesion Molecules / genetics
  • Cell Adhesion Molecules / physiology*
  • Cell Line
  • Cell Polarity / genetics*
  • Cell Polarity / physiology*
  • Cytoplasm / metabolism
  • Flow Cytometry
  • Fluorescent Antibody Technique
  • Hepatocytes / physiology*
  • Humans
  • Immunoglobulins / genetics
  • Immunoglobulins / physiology*
  • Lentivirus / genetics
  • Molecular Sequence Data
  • Phosphorylation
  • Plasmids / genetics
  • Protein Kinase C / metabolism
  • Rats
  • Receptors, Cell Surface
  • Threonine / metabolism
  • Transduction, Genetic

Substances

  • Cell Adhesion Molecules
  • F11R protein, human
  • Immunoglobulins
  • Receptors, Cell Surface
  • Threonine
  • Protein Kinase C