JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells.
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.
Braiterman
Lelita T
LT
Department of Cell Biology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205, USA. lbraite1@jhmi.edu
Heffernan
Sean
S
Nyasae
Lydia
L
Johns
David
D
See
Alfred P
AP
Yutzy
Rebeca
R
McNickle
Allison
A
Herman
Mira
M
Sharma
Arun
A
Naik
Ulhas P
UP
Hubbard
Ann L
AL
D000595:Amino Acid Sequence; D000818:Animals; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D016764:Cell Polarity / Q000235:genetics* / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D022781:Hepatocytes / Q000502:physiology*; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface; D013912:Threonine / Q000378:metabolism; D014161:Transduction, Genetic
D015815:Cell Adhesion Molecules; C407243:F11R protein, human; D007136:Immunoglobulins; D011956:Receptors, Cell Surface; D013912:Threonine; D011493:Protein Kinase C