Evidence for ERK1/2 phosphorylation controlling contact inhibition of proliferation in Madin-Darby canine kidney epithelial cells

Li, Shixiong; Gerrard, Edward R.; Balkovetz, Daniel F.

doi:10.1152/ajpcell.00020.2004

Cited by 28 publications

(32 citation statements)

References 38 publications

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“…DNA laddering experiment showed that apoptosis was induced by serum starvation in both MDCK and b-KD cells indicating that reduced Na,K-b 1 expression does not protect cells from apoptosis. ERK1/2 phosphorylation has been shown to regulate contact inhibition of proliferation in MDCK cells grown in 2D culture (Li et al, 2004). Our results are consistent these findings in 2D culture.…”

Section: Discussionsupporting

confidence: 92%

“…S4), suggesting that knockdown of Na,K-b 1 did not protect the cells from apoptosis under serum starvation and that reduced apoptosis is likely to play a minimal role in lumen filling in b-KD cells. ERK1/2 activation is known to suppress contact inhibition of cell proliferation in MDCK cells and its phosphorylation levels decline as cells attain higher densities (Li et al, 2004). We hypothesized that increased ERK1/2 levels in b-KD cells are associated with enhanced cell proliferation and suppression of contact inhibition.…”

Section: Resultsmentioning

confidence: 97%

“…Cells were plated at high densities on Transwell filters (0.4 mm pore size) and allowed to grow and polarize for three days as described previously (Li et al, 2004).…”

Section: Immunoblottingmentioning

confidence: 99%

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Na,K-ATPase β-subunit cis homo-oligomerization is necessary for epithelial lumen formation in mammalian cells

Barwe

Skay

McSpadden

et al. 2012

Journal of Cell Science

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SummaryNa,K-ATPase is a hetero-oligomer of an a-and a b-subunit. The a-subunit (Na,K-a) possesses the catalytic function, whereas the bsubunit (Na,K-b) has cell-cell adhesion function and is localized to the apical junctional complex in polarized epithelial cells. Earlier, we identified two distinct conserved motifs on the Na,K-b 1 transmembrane domain that mediate protein-protein interactions: a glycine zipper motif involved in the cis homo-oligomerization of Na,K-b 1 and a heptad repeat motif that is involved in the hetero-oligomeric interaction with Na,K-a 1 . We now provide evidence that knockdown of Na,K-b 1 prevents lumen formation and induces activation of extracellular regulated kinases 1 and 2 (ERK1/2) mediated by phosphatidylinositol 3-kinase in MDCK cells grown in three-dimensional collagen cultures. These cells sustained cell proliferation in an ERK1/2-dependent manner and did not show contact inhibition at high cell densities, as revealed by parental MDCK cells. This phenotype could be rescued by wild-type Na,K-b 1 or heptad repeat motif mutant of Na,K-b 1 , but not by the glycine zipper motif mutant that abrogates Na,K-b 1 cis homo-oligomerization. These studies suggest that Na,K-b 1 cis homo-oligomerization rather than hetero-oligomerization with Na,K-a 1 is involved in epithelial lumen formation. The relevance of these findings to pre-neoplastic lumen filling in epithelial cancer is discussed.

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Section: Discussionsupporting

confidence: 92%

Section: Resultsmentioning

confidence: 97%

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Na,K-ATPase β-subunit cis homo-oligomerization is necessary for epithelial lumen formation in mammalian cells

Barwe

Skay

McSpadden

et al. 2012

Journal of Cell Science

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“…Activity of ERK has previously been linked to tubular cell proliferation (16), and ERK is upregulated in the Han:SPRD rat model of ADPKD (20). Furthermore, both ATP and UTP are potent activators of ERK in human intestine (38), and P2Y receptor-mediated activation of ERK via a cAMP-dependent pathway has also been shown to increase proliferation of dendritic cells (21).…”

Section: Discussionmentioning

confidence: 98%

Antagonism of endogenous putative P2Y receptors reduces the growth of MDCK-derived cysts cultured in vitro

Turner¹,

King²,

Srai³

et al. 2007

American Journal of Physiology-Renal Physiology

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Turner CM, King BF, Srai KS, Unwin RJ. Antagonism of endogenous putative P2Y receptors reduces the growth of MDCK-derived cysts cultured in vitro. Am J Physiol Renal Physiol 292: F15-F25, 2007. First published July 18, 2006; doi:10.1152/ajprenal.00103.2006.-P2Y receptors couple to G proteins and either mobilize intracellular Ca 2ϩ or alter cAMP levels to modulate the activity of Ca 2ϩ -and cAMPsensitive ion channels. We hypothesize that increased ion transport into the lumen of MDCK cysts can osmotically drive fluid movement and increase cyst size. Furthermore, activation of the adenylate cyclase/cAMP pathway may trigger cell proliferation via an extracellular signal-related kinase cascade. To test this hypothesis, several P2Y receptor inhibitors were used on the MDCK in vitro model of renal cyst formation. The nonspecific P2 receptor inhibitors reactive blue 2 and suramin reduced cyst growth significantly, as did PPADS and, to a lesser extent, the P2Y1-specific antagonist MRS2179. Cyst growth was reduced by ϳ50% when ATP was removed from the culture medium with apyrase, although stable analogs of ATP failed to increase cyst size. The nonselective P2X receptor inhibitor Coomassie brilliant blue G was ineffective at reducing cyst growth, suggesting no involvement of P2X receptors. Finally, the presence of selective inhibitors of ERK activation (either PD98059 or U0126) greatly reduced cyst growth, whereas in untreated cysts ERK activity was observed to increase with time. We conclude that stimulation of endogenous P2Y receptors by extracellular ATP increases growth of MDCK cysts via cAMP-dependent activation of the ERK pathway. P2Y receptor antagonists may have therapeutic potential in reducing cyst size and slowing disease progression; although further studies in vitro and in vivo are needed to investigate the specificity and role of these P2Y receptors in renal cystic diseases. ATP; polycystic kidney disease; Madin-Darby canine kidney; extracellular signal related kinase; renal cyst growth; ADPKD AUTOSOMAL DOMINANT POLYCYSTIC kidney disease (ADPKD) affects 1 in 1,000 births and accounts for 5% of hemodialysis patients worldwide. Most cases of ADPKD are caused by loss-of-function mutations in the PKD1 and PKD2 genes that encode the proteins polycystin-1 (TRPP1) and polcystin-2 (TRPP2), respectively. The disease is characterized by the progressive development and enlargement of multiple cysts in both kidneys and is the most common inherited cause of renal failure.During cystogenesis, the normal and mainly reabsorptive role of the renal epithelium is reversed to a predominantly secretory one, although the mechanisms underlying this change are poorly understood. However, it is generally thought that cyst expansion is the result of proliferation of cyst wall epithelial cells and accumulation of fluid within the cyst cavity. It is known that the rate of ATP-release is elevated in isolated epithelial cells cultured from human and mouse polycystic kidneys and that the ATP concentration in cyst luminal fluid is signific...

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“…C ells characteristically form epithelial monolayers through logarithmic growth when cells are subconfluent followed by cellto-cell contact and concluding with contact inhibition of proliferation (CIP), proliferative quiescence, and epithelial monolayer maturation, including tight junction (TJ) formation (1,2). CIP is an important step in monolayer maturation that is mediated in part by the activation of the Hippo pathway.…”

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confidence: 99%

Occludin S471 Phosphorylation Contributes to Epithelial Monolayer Maturation

Bolinger

Ramshekar

Waldschmidt

et al. 2016

Molecular and Cellular Biology

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Multiple organ systems require epithelial barriers for normal function, and barrier loss is a hallmark of diseases ranging from inflammation to epithelial cancers. However, the molecular processes regulating epithelial barrier maturation are not fully elucidated. After contact, epithelial cells undergo size-reductive proliferation and differentiate, creating a dense, highly ordered monolayer with high resistance barriers. We provide evidence that the tight junction protein occludin contributes to the regulation of epithelial cell maturation upon phosphorylation of S471 in its coiled-coil domain. Overexpression of a phosphoinhibitory occludin S471A mutant prevents size-reductive proliferation and subsequent tight junction maturation in a dominant manner. Inhibition of cell proliferation in cell-contacted but immature monolayers recapitulated this phenotype. A kinase screen identified G-protein-coupled receptor kinases (GRKs) targeting S471, and GRK inhibitors delayed epithelial packing and junction maturation. We conclude that occludin contributes to the regulation of size-reductive proliferation and epithelial cell maturation in a phosphorylation-dependent manner.

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Evidence for ERK1/2 phosphorylation controlling contact inhibition of proliferation in Madin-Darby canine kidney epithelial cells

Cited by 28 publications

References 38 publications

Na,K-ATPase β-subunit cis homo-oligomerization is necessary for epithelial lumen formation in mammalian cells

Na,K-ATPase β-subunit cis homo-oligomerization is necessary for epithelial lumen formation in mammalian cells

Antagonism of endogenous putative P2Y receptors reduces the growth of MDCK-derived cysts cultured in vitro

Occludin S471 Phosphorylation Contributes to Epithelial Monolayer Maturation

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