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

Lelita T. Braiterman, Sean Heffernan, Lydia Nyasae, David Johns, Alfred P. See, Rebeca Yutzy, Allison McNickle, Mira Herman, Arun Sharma, Ulhas P. Naik, Ann L. Hubbard

Research output: Contribution to journalArticle

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 ∼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 (huΔC-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, huΔC-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.

Original languageEnglish (US)
Pages (from-to)G576-G588
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume294
Issue number2
DOIs
StatePublished - Feb 1 2008

Keywords

  • Partitioning-defective polarity protein/atypical protein kinase C complex

ASJC Scopus subject areas

  • Physiology
  • Hepatology
  • Gastroenterology
  • Physiology (medical)

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    Braiterman, L. T., Heffernan, S., Nyasae, L., Johns, D., See, A. P., Yutzy, R., McNickle, A., Herman, M., Sharma, A., Naik, U. P., & Hubbard, A. L. (2008). JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells. American Journal of Physiology - Gastrointestinal and Liver Physiology, 294(2), G576-G588. https://doi.org/10.1152/ajpgi.00159.2007