The studies presented in this paper demonstrate that distinct signals are required to mediate RGC neurite outgrowth on the classical cadherins E-, N-, and R-cadherin. Our results are summarized in a model presented in . The first aspect of the model that should be considered is the mode of recognition between the cadherin molecules on the substrate and the RGC neurites. In general, classical cadherins are homophilic cell-cell adhesion molecules (Halbleib and Nelson, 2006
). However, heterophilic interactions of cadherins have been observed (Duguay et al., 2003
; Foty and Steinberg, 2005
; Niessen and Gumbiner, 2002
). In our model, we suggest that both N- and E-cadherin interact homophilically to mediate outgrowth, but that R-cadherin may interact heterophilically with N-cadherin to mediate outgrowth (). This assertion is supported by the fact that when L-cells, which do not express endogenous cadherins and do not spontaneously aggregate, are transfected with cDNA encoding N-cadherin or R-cadherin and are mixed together in an aggregation assay they form chimeric aggregates (Inuzuka et al., 1991a
; Matsunami et al., 1993
), demonstrating that R-cadherin can interact in trans
with N-cadherin. Our experiments show that R-cadherin-mediated outgrowth is blocked by the addition of the chick-specific N-cadherin function-blocking antibody, NCD-2. This demonstrates that R-cadherin-mediated neurite outgrowth requires N-cadherin molecules on the RGC neurite, potentially implicating a heterophilic R-cadherin/N-cadherin interaction (). It is also possible that R-cadherin-mediated neurite outgrowth is homophilic but requires cis
interactions with N-cadherin on the RGC neurite (Shan et al., 2000
) (see ).
Reversible tyrosine phosphorylation of cadherins serves as a key regulatory mechanism for cadherin-mediated adhesion. For example, tyrosine phosphorylation of the intracellular segment of the classical cadherins results in a loss of cadherin-mediated adhesion and destabilization of adherens junctions (Sallee et al., 2006
; Yap et al., 2007
). Dephosphorylation of the classical cadherins or their associated proteins has also been shown to regulate cell adhesion. The tyrosine phosphatase PTPμ associates with E-, N- and R-cadherin (Brady-Kalnay et al., 1998
; Brady-Kalnay et al., 1995
) and is required for E-cadherin-mediated cell adhesion (Hellberg et al., 2002
). Within the chick retina, RGCs express PTPμ during axon extension (Burden-Gulley and Brady-Kalnay, 1999
). Our laboratory has previously demonstrated that PTPμ expression and catalytic activity are required for both E- and N-cadherin-mediated neurite outgrowth (Burden-Gulley and Brady-Kalnay, 1999
; Oblander et al., 2007
). In the current study, we demonstrate that PTPμ activity is also required for R-cadherin-mediated neurite outgrowth. We hypothesize that PTPμ alters cadherin function by recruiting different regulatory molecules to the cadherin/catenin complex thus distinctly regulating cadherin-mediated adhesion/neurite outgrowth ().
The Rho GTPase subfamily members are likely candidates for signaling molecules that may be recruited by PTPμ to regulate cadherin-mediated outgrowth. Cdc42, Rac1 and RhoA play a central role in regulating cell adhesion through dynamic rearrangement of the cytoskeleton, reviewed in (Heasman and Ridley (2008)
). Rho GTPases have also been implicated in regulating axon guidance and neurite outgrowth (Koh, 2006
; Linseman and Loucks, 2008
). Activation of Cdc42 induces filopodia formation while Rac1 activation promotes lamellipodia formation. One means of regulating the Rho GTPases is through binding to cytoplasmic proteins that stabilize the active forms of the proteins or that recruit GTPases to their substrates. IQGAP1 is an example of one such protein. IQGAP1 interacts with activated Rac1 and Cdc42 (Briggs and Sacks, 2003
). IQGAP1 has a higher affinity for Cdc42 than Rac1 and acts to stabilize GTP-bound Cdc42 (Erickson et al., 1997
; Hart et al., 1996
; Kuroda et al., 1998
; Swart-Mataraza et al., 2002
; Zhang et al., 1997
In this manuscript, we determined that Rac1 activity is required for E-, N-, and R-cadherin-mediated neurite outgrowth. The Rac1 inhibitor that was used blocks the interaction of Rac1 with its GEFs Trio and Tiam-1 (Gao et al., 2004
). Trio is a GEF that binds the RPTP LAR (Debant et al., 1996
). Tiam-1 is known to regulate cadherin-dependent adhesion (Kraemer et al., 2007
; Malliri et al., 2004
). Future studies will investigate Trio and Tiam-1 binding to PTPμ and the cadherins in the retina. Our results also identified a requirement for Cdc42 activity in N- and R-cadherin-mediated neurite outgrowth. Using an IQGAP1 inhibitor peptide that competes for binding to the Cdc42 and Rac1 binding site on IQGAP1, we demonstrated that an interaction between Cdc42/Rac1 and IQGAP1 likely contributes to N- and R-cadherin-mediated neurite outgrowth. We hypothesize that PTPμ recruits IQGAP1/Cdc42 to N- and R- cadherin to regulate neurite outgrowth (). IQGAP1 binds β-catenin, which displaces cadherin from binding β-catenin and downregulates cell adhesion (Fukata et al., 1999
). Our hypothesis is that IQGAP1 binding to PTPμ would prevent IQGAP1 binding to β-catenin, which may stabilize cadherin-dependent cell adhesion. IQGAP1 binding to PTPμ is enhanced in the presence of Cdc42 (Phillips-Mason et al., 2006
), therefore, IQGAP1 would likely couple PTPμ to Cdc42. Because IQGAP1 was not required for E-cadherin-mediated retinal neurite outgrowth we suggest that Rac1 is recruited to E-cadherin via an alternative mechanism ().
It is important to consider whether cross-talk between the Rho GTPases may be occurring in our study of the regulation of cadherin-mediated neurite outgrowth. We previously examined whether inhibiting one Rho GTPase in neurite outgrowth assays may be having unintended secondary effects on other Rho GTPases (Major and Brady-Kalnay, 2007
). Using different combinations of DN-Rho GTPases we demonstrated that there is no effect of combination therapy versus single treatment of a DN-Rho GTPase on PTPμ-mediated neurite outgrowth (Major and Brady-Kalnay, 2007
). We predict therefore that alteration of neurite outgrowth observed in our present study is due to the addition of the DN-Rho GTPases and not secondary effects caused by changes in activation of other Rho GTPases. In addition, the Rac1 specific inhibitor has no effect on Cdc42 or RhoA (Gao et al., 2004
). Furthermore, each Rho GTPase exhibited distinct effects on each cadherin substrate.
We hypothesize that PTPμ also plays a role in recruiting the protein kinase C (PKC) family of ipid-dependent serine-threonine kinases to regulate cadherin-mediated neurite outgrowth. PKCs have been implicated in the regulation of E-cadherin-mediated adhesion and formation of adherens junctions (Hellberg et al., 2002
; Lewis et al., 1994
; Skoudy and Garcia de Herreros, 1995
). Using the PKCδ inhibitor Rottlerin, we demonstrate here that PKCδ serine-threonine kinase signaling is required for E- and R-cadherin-mediated neurite outgrowth but not N-cadherin-mediated neurite outgrowth. Furthermore, we show that R-cadherin-mediated neurite outgrowth may require additional PKCs.
Rottlerin was originally described as a PKC inhibitor with specificity for PKCδ (Gschwendt et al., 1994
). After this initial publication, it was reported that Rottlerin is ineffective at directly inhibiting PKCδ activity but likely inhibits the enzyme by an indirect mechanism via ATP depletion, reactive oxygen species generation and altering PKCδ tyrosine phosphorylation (Soltoff, 2007
). Additional studies have demonstrated, however, that Rottlerin does decrease PKCδ activity (Chaudhuri et al., 2005
; Fan et al., 2006
; Fan et al., 2009
). In addition, treatment of various neuronal cell types with Rottlerin results in data comparable to that of the same neuronal cell types treated with dominant negative PKCδ (Zhang et al., 2007
), neurons from PKCδ knockout mice (Zhang et al., 2007
; Zhang et al., 2009
), PKCδ-small interfering RNA (Zhang et al., 2007
) or catalytically inactive PKCδK376R (Kanthasamy et al., 2008
) supporting the assertion that Rottlerin inhibits PKCδ activity.
Activated PKC binds to the scaffolding protein RACK1 and is then recruited to the plasma membrane where it can phosphorylate its substrates (Sklan et al., 2006
). PTPμ is found in a complex with RACK1 (Mourton et al., 2001
) and PKCδ in cultured retinal cells (RNEs) and in E8 retinal tissue (Rosdahl et al., 2002
). PKCδ activity and the interaction between PKCδ and RACK1 are required for PTPμ-mediated neurite outgrowth (Ensslen and Brady-Kalnay, 2004
; Rosdahl et al., 2002
). Furthermore, a multimolecular complex consisting of PKCβII
, RACK1, PTPμ, β-catenin, and E-cadherin among other proteins was identified in mouse kidney collecting duct epithelial cells (Chattopadhyay et al., 2003
). Disruption of the PKCβII
/RACK1 interaction blocked the association of the multimolecular complex with α-actinin and resulted in a decrease in cell-cell adhesion, demonstrating that PKCβII
is required to stabilize the PTPμ/E-cadherin/β-catenin complex with the actin cytoskeleton. We therefore hypothesize that PTPμ/RACK1/PKCδ is recruited to the cadherin/catenin complex at the cell membrane where PKCδ may regulate E- or R-cadherin-dependent processes, but not N-cadherin-dependent neurite outgrowth ().
The difference in the ability of retinal neurites from the dorsal-nasal quadrant of the retina to grow on a substrate of R- but not N-cadherin warrants further examination. We speculate that in this retinal quadrant the signals required for R-cadherin outgrowth are present whereas those required for N-cadherin outgrowth are missing. Those signals could be at the level of the receptor or within the neurites. Considering the former, perhaps the levels of N-cadherin receptor expressed on the surface of the RGCs are too low in this retinal quadrant for homophilic trans
interactions to mediate N-cadherin-dependent neurite outgrowth, but is sufficient to act in concert either in cis
with R-cadherin on RGCs to mediate outgrowth on R-cadherin. The absence of a chick R-cadherin specific function-blocking antibody precludes us from testing this hypothesis at the moment. Alternatively, yet unidentified signals downstream of N-cadherin may be lacking in the dorsal-nasal retina that are not required for R- and E-cadherin-mediated outgrowth. Our current study supports this hypothesis given that the signals required for neurite outgrowth are not the same for all three cadherins. Furthermore, differential signaling via PKCδ in the nasal and temporal retina has been demonstrated to explain the differential responses of RGC neurites to a substrate of PTPμ (Ensslen and Brady-Kalnay, 2004
). Future studies that identify distinct N-cadherin signals may help test these hypotheses.
To summarize, the studies we present in this manuscript demonstrate that distinct signal transduction pathways are required for neurite outgrowth mediated by individual classical cadherins. We hypothesize that PTPμ recruits different adapter proteins to distinct cadherin complexes to transduce signals on that particular substrate. For E-cadherin-mediated neurite outgrowth, PTPμ, PKCδ, and Rac1 are required (). For N-cadherin-mediated neurite outgrowth, PTPμ, Rac1, and Cdc42 activities, but not PKC activity are required (). Finally, for R-cadherin-mediated neurite outgrowth, PTPμ, Rac1, Cdc42, and PKCs are required (). We hypothesize that the differences we observed in growth cone morphology on the purified cadherins can be at least partially explained by these distinct signaling pathways. Future studies will also determine whether the above-mentioned signaling pathways regulate cadherin-dependent outgrowth via inside-out or outside-in mechanisms.