Patrick Laprise

Down-regulation of MEK/ERK signaling by E-cadherin-dependent PI3K/Akt pathway in differentiating intestinal epithelial cells

In vitro experiments have shown that the establishment of cell–cell contacts in intestinal epithelial cell cultures is a critical step in initiating ERK inhibition, cell cycle arrest, and induction of the differentiation process. Herein, we determined the mechanisms through which E‐cadherin‐mediated cell–cell contacts modulate the ERK pathway in intestinal epithelial cells. We report that: (1) removal of calcium from the culture medium of newly confluent Caco‐2/15 cells (30 min, 4 mM EGTA) results in the disruption of both adherens and tight junctions and clearly decreases Akt phosphorylation while increasing MEK and ERK activities. Akt, MEK, and ERK activation levels return to control levels 60 min after calcium restoration; (2) the use of E‐cadherin blocking antibodies efficiently prevents Akt phosphorylation and MEK–ERK inhibition after 70 min of calcium restoration; (3) using the PI3K inhibitor LY294002 (15 μM) in calcium switch experiments, we demonstrate that the assembly of adherens junctions activates Akt activity and triggers the inhibition of ERK1/2 activities in a PI3K‐dependent manner; (4) adenoviral infection of confluent Caco‐2/15 cells with a constitutively active mutant of Akt1 strongly represses ERK1/2 activities; (5) inhibition of PI3K abolishes Akt activity but leads to a rapid and sustained activation of the MEK–ERK1/2 in confluent differentiating Caco‐2/15 cells, but not in undifferentiated growing Caco‐2/15 cells. Our data suggest that E‐cadherin engagement leads to MEK/ERK inhibition in a PI3K/Akt‐dependent pathway. This mechanism may account for the role of E‐cadherin in proliferation/differentiation transition along the crypt‐villus axis of the human intestinal epithelium. J. Cell. Physiol. 199: 32–39, 2004© 2003 Wiley‐Liss, Inc.

Merosin‐integrin promotion of skeletal myofiber cell survival: Differentiation state‐distinct involvement of p60Fyn tyrosine kinase and p38α stress‐activated MAP kinase

Myofiber survival and suppression of anoikis depend in large part on the merosin (laminin‐2/‐4)‐integrin α7β1D cell adhesion system; however, the question remains as to the nature of the signaling molecules/pathways involved. In the present study, we investigated this question using the C2C12 cell model of myogenic differentiation and its merosin‐ and laminin‐deficient derivatives. Herein, we report that: 1) of four members of the Src family of tyrosine kinases studied (p60Src, p53/56Lyn, p59Yes, or p60Fyn), the expression and activity of p60Fyn are found in myotubes exclusively; 2) a severe decrease of p60Fyn activity correlates with myotube apoptosis/anoikis induced by pharmocological compounds (herbimycin A or PP2) which inhibit tyrosine kinases of the Src family, by merosin deficiency and by β1 integrin inhibition; 3) myoblast survival depends on Fak and the MEK/Erk pathway, in contrast to myotubes; 4) the PI3‐K pathway is not involved in either myoblast or myotube survival; and 5) p38α SAPK stimulation and activity (but not that of p38β) are required in the progression of myotube apoptosis/anoikis induced by p60Fyn inhibition, merosin deficiency or β1 integrin‐inhibition; however, p38 is not involved in myoblast apoptosis. Taken together, these results suggest that the promotion of myotube survival by the merosin‐α7β1D adhesion system involves p60Fyn, and that disruptions in this cell adhesion system induce myotube apoptosis/anoikis through a p38α SAPK‐dependent pathway. J. Cell. Physiol. 191: 69–81, 2002.

Differential sensitivity to apoptosis between the human small and large intestinal mucosae: linkage with segment-specific regulation of BCL-2 homologs and involvement of signaling pathways.

The small and large intestines differ in their expression profiles of Bcl‐2 homologs. Intestinal segment‐specific Bcl‐2 homolog expression profiles are acquired as early as by mid‐gestation (18–20 weeks) in man. In the present study, we examined the question whether such distinctions underlie segment‐specific control mechanisms of intestinal cell survival. Using mid‐gestation human jejunum and colon organotypic cultures, we analyzed the impact of growth factors (namely insulin; 10 μg/ml) and pharmacological compounds that inhibit signal transduction molecules/pathways (namely tyrosine kinases, Fak, PI3‐K/Akt, and MEK/Erk) on cell survival and Bcl‐2 homolog expression (anti‐apoptotic: Bcl‐2, Bcl‐XL, Mcl‐1; pro‐apoptotic: Bax, Bak, Bad). The relative activation levels of p125Fak, p42Erk‐2, and p57Akt were analyzed as well. Herein, we report that (1) the inhibition of signal transduction molecules/pathways revealed striking differences in their impact on cell survival in the jejunum and colon (e.g., the inhibition of p125Fak induced apoptosis with a significantly greater extent in the jejunum [∼43%] than in the colon [∼24%]); (2) sharp distinctions between the two segments were noted in the modulatory effects of the various treatments on Bcl‐2 homolog steady‐state levels (e.g., inhibition of tyrosine kinase activities in the jejunum down‐regulated all anti‐apoptotics analyzed while increasing Bax, whereas the same treatment in the colon down‐regulated Bcl‐XL only and increased all pro‐apoptotics); and (3) in addition to their differential impact on cell survival and Bcl‐2 homolog expression, the MEK/Erk and PI3‐K/Akt pathways were found to be distinctively regulated in the jejunum and colon mucosae (e.g., insulin in the jejunum increased p42Erk‐2 activation without affecting that of p57Akt, whereas the same treatment in the colon decreased p42Erk‐2 activation while increasing that of p57Akt). Altogether, these data show that intestinal cell survival is characterized by segment‐specific susceptibilities to apoptosis, which are in turn linked with segmental distinctions in the involvement of signaling pathways and the regulation of Bcl‐2 homolog steady‐state levels. Therefore, these indicate that cell survival is subject to segment‐specific control mechanisms along the proximal‐distal axis of the intestine. J. Cell. Biochem. 82: 339–355, 2001.

Cyclic AMP-dependent protein kinase A negatively modulates adherens junction integrity and differentiation of intestinal epithelial cells

Intestinal epithelial cell differentiation is a complex process in which many different signaling pathways are likely involved. An increase in the intracellular levels of cyclic AMP (cAMP) has been shown to inhibit enterocyte differentiation; however, the mechanisms through which cAMP/PKA signaling modulates differentiation of human intestinal epithelial cells are still not well understood. Herein, we report that: (1) treatment of Caco‐2/15 cells with 8Br‐cAMP repressed sucrase–isomaltase and villin protein expression and strongly attenuated morphological differentiation of enterocyte‐like features in Caco‐2/15 such as epithelial cell polarity and brush border formation; (2) treatment of confluent Caco‐2/15 cells with 8Br‐cAMP led to a strong decrease in F‐actin localized at cell–cell contact sites along with a reduced amount of E‐cadherin and catenins, but not of ZO‐1, at cell–cell interfaces concomitant with a decreased association of these proteins with the actin cytoskeleton; (3) inhibition of PKA by H89 prevented disruption of adherens junctions by extracellular calcium depletion; (4) treatment of Caco‐2/15 cells with 8Br‐cAMP prevented the recruitment and activation of p85/PI‐3K to E‐cadherin‐mediated cell–cell contacts, an important event in the assembly of adherens junctions and differentiation of these cells; (5) E‐cadherin appears to be phosphorylated on serine in vivo in a PKA‐dependent mechanism. Conclusion: Our studies show that cAMP/PKA signaling negatively regulates adherens junction integrity as well as morphological and functional differentiation of intestinal epithelial cells.

Merosin (laminin‐2/4)‐driven survival signaling: Complex modulations of Bcl‐2 homologs

We have shown previously that the promotion of myofiber survival by the basement membrane component merosin (laminin‐2 [α2β1γ1]/laminin‐4 [α2β2γ1]) is dependent on the activity of the tyrosine kinase Fyn, whereas myofiber anoikis induced by merosin deficiency is dependent on the stress‐activated protein kinase p38α. To further understand such merosin‐driven survival signaling, we analyzed the expression of five Bcl‐2 homologs (Bcl‐2, Bcl‐XL, Bax, Bak, Bad) and one non‐homologous associated molecule (Bag‐1) in normal and merosin‐deficient myotubes, with or without pharmacological inhibitors for Fyn and p38. Herein, we report that (1) merosin deficiency induces anoikis and causes decreased Bcl‐2, Bcl‐XL, and Bag‐1 levels, increased Bax and Bak levels, and decreased Bad phosphorylation; (2) Bcl‐2, Bcl‐XL, Bag‐1, and Bad phosphorylation are also decreased in anoikis‐dying, Fyn‐inhibited myotubes; (3) the inhibition of p38α in Fyn‐inhibited and/or merosin‐deficient myotubes protects against anoikis and increases Bcl‐2 levels above normal, in addition to restoring Bad phosphorylation and Bag‐1 levels to normal; (4) the overexpression of merosin in deficient myotubes also rescues from anoikis and increases Bcl‐2 levels and Bad phosphorylation above normal, in addition to restoring Bcl‐XL, Bag‐1, Bax, and Bak levels to normal; and (5) Bcl‐2 overexpression is sufficient to rescue merosin‐deficient myotubes from anoikis, even though the expression/phosphorylation levels of the other homologs analyzed are not restored to normal. These results indicate that merosin‐driven myofiber survival signaling affects complex, differential modulations of individual Bcl‐2 homologs. These further suggest that Bcl‐2 can play a major role in suppressing myofiber anoikis.