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Collagen I-mediated up-regulation of N-cadherin requires cooperative signals from integrins and discoidin domain receptor 1.

Shintani Y, Fukumoto Y, Chaika N, Svoboda R, Wheelock MJ, Johnson KR - J. Cell Biol. (2008)

Bottom Line: Focal adhesion kinase (FAK)-related protein tyrosine kinase (Pyk2) is downstream of DDR1, whereas FAK is downstream of alpha2beta1 integrin.Both receptor complexes rely on the p130 Crk-associated substrate scaffold.Interestingly, Rap1, but not Rho family guanosine triphosphatases, is required for the response to collagen I.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.

ABSTRACT
Tumor cells undergo epithelial-to-mesenchymal transition (EMT) to convert from a benign to a malignant phenotype. Our recent focus has been signaling pathways that promote EMT in response to collagen. We have shown that human pancreatic cancer cells respond to collagen by up-regulating N-cadherin, which promotes tumor growth, invasion, and metastasis. Initial characterization showed that knocking down c-Jun NH2-terminal kinase prevented N-cadherin up-regulation and limited tumor growth and invasion in a mouse model for pancreatic cancer. The current study was designed to understand the pathway from collagen to N-cadherin up-regulation. Initiation of the signal requires two collagen receptors, alpha2beta1 integrin and discoidin domain receptor (DDR) 1. Each receptor propagates signals through separate pathways that converge to up-regulate N-cadherin. Focal adhesion kinase (FAK)-related protein tyrosine kinase (Pyk2) is downstream of DDR1, whereas FAK is downstream of alpha2beta1 integrin. Both receptor complexes rely on the p130 Crk-associated substrate scaffold. Interestingly, Rap1, but not Rho family guanosine triphosphatases, is required for the response to collagen I.

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JNK1/cJun activation plays a crucial role in collagen I–mediated changes in BxPC3 cells. (A) Mock BxPC3 cells or cells expressing MKK7-JNK1 (constitutively active JNK1) or MKK7-KM-JNK1 (inactive control) were extracted 2 d after plating on noncoated or collagen I–coated dishes. 30 or 60 μg of protein was blotted for N-cadherin, HA tag (to detect MKK7/JNK1), or tubulin. (B) BxPC3 cells expressing MKK7-JNK1 (a and b) or MKK7-KM-JNK1 (c and d) were cultured on noncoated (a and c) or collagen I–coated (b and d) dishes for 2 d. Bar, 100 μm. (C) 30 μg of protein from mock BxPC3 cells or cells expressing dominant-negative cJun (cJun-DN) were extracted 2 d after plating on noncoated or collagen I–coated dishes, resolved by SDS-PAGE, and blotted for N-cadherin, total cJun, or tubulin. (D) Mock BxPC3 cells (a and b) or cells expressing cJun-DN (c and d) were cultured on noncoated (a and c) or collagen I–coated (b and d) dishes for 2 d. Bar, 100 μm.
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fig8: JNK1/cJun activation plays a crucial role in collagen I–mediated changes in BxPC3 cells. (A) Mock BxPC3 cells or cells expressing MKK7-JNK1 (constitutively active JNK1) or MKK7-KM-JNK1 (inactive control) were extracted 2 d after plating on noncoated or collagen I–coated dishes. 30 or 60 μg of protein was blotted for N-cadherin, HA tag (to detect MKK7/JNK1), or tubulin. (B) BxPC3 cells expressing MKK7-JNK1 (a and b) or MKK7-KM-JNK1 (c and d) were cultured on noncoated (a and c) or collagen I–coated (b and d) dishes for 2 d. Bar, 100 μm. (C) 30 μg of protein from mock BxPC3 cells or cells expressing dominant-negative cJun (cJun-DN) were extracted 2 d after plating on noncoated or collagen I–coated dishes, resolved by SDS-PAGE, and blotted for N-cadherin, total cJun, or tubulin. (D) Mock BxPC3 cells (a and b) or cells expressing cJun-DN (c and d) were cultured on noncoated (a and c) or collagen I–coated (b and d) dishes for 2 d. Bar, 100 μm.

Mentions: Thus far, we have characterized a novel signaling pathway initiated by interactions with collagen I that requires both integrin and DDR1, is propagated through FAK and Pyk2, requires the p130CAS scaffold, and requires Rap1 GTPase. The outcome is activation of JNK1, using the upstream kinases MKK7 and MLK3. To confirm that activated JNK1 up-regulates expression of N-cadherin, we expressed constitutively active JNK1 in BxPC3 cells. It has been reported that fusing MKK7 to JNK1 results in constitutive JNK1 activity and activates c-Jun in the absence of any stimulus (Zheng et al., 1999). The JNK1 portion of the fusion protein is phosphorylated on both Thr183 and Tyr185 residues by the MKK7 portion of the fusion protein. Overexpression of MKK7-JNK1 increased N-cadherin expression even when cells were plated on noncoated dishes, whereas a control construct with kinase-dead MKK7 did not, showing that activation of JNK is sufficient to up-regulate N-cadherin (Fig. 8). Interestingly, overexpression of MKK7-JNK1 did not induce cell scattering on noncoated dishes, indicating that although JNK1 activation is necessary for cell scattering, it is not sufficient to induce cell scattering in the absence of a collagen I stimulus (Fig. 5 B). These data are consistent with our previous studies (Shintani et al., 2006a) and with data in Fig. S5 (available at http://www.jcb.org/cgi/content/full/jcb.200708137/DC1) showing that artificially up-regulating N-cadherin also did not induce cell scattering in the absence of a collagen I signal, indicating that up-regulation of N-cadherin is necessary, but not sufficient, for cell scattering.


Collagen I-mediated up-regulation of N-cadherin requires cooperative signals from integrins and discoidin domain receptor 1.

Shintani Y, Fukumoto Y, Chaika N, Svoboda R, Wheelock MJ, Johnson KR - J. Cell Biol. (2008)

JNK1/cJun activation plays a crucial role in collagen I–mediated changes in BxPC3 cells. (A) Mock BxPC3 cells or cells expressing MKK7-JNK1 (constitutively active JNK1) or MKK7-KM-JNK1 (inactive control) were extracted 2 d after plating on noncoated or collagen I–coated dishes. 30 or 60 μg of protein was blotted for N-cadherin, HA tag (to detect MKK7/JNK1), or tubulin. (B) BxPC3 cells expressing MKK7-JNK1 (a and b) or MKK7-KM-JNK1 (c and d) were cultured on noncoated (a and c) or collagen I–coated (b and d) dishes for 2 d. Bar, 100 μm. (C) 30 μg of protein from mock BxPC3 cells or cells expressing dominant-negative cJun (cJun-DN) were extracted 2 d after plating on noncoated or collagen I–coated dishes, resolved by SDS-PAGE, and blotted for N-cadherin, total cJun, or tubulin. (D) Mock BxPC3 cells (a and b) or cells expressing cJun-DN (c and d) were cultured on noncoated (a and c) or collagen I–coated (b and d) dishes for 2 d. Bar, 100 μm.
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fig8: JNK1/cJun activation plays a crucial role in collagen I–mediated changes in BxPC3 cells. (A) Mock BxPC3 cells or cells expressing MKK7-JNK1 (constitutively active JNK1) or MKK7-KM-JNK1 (inactive control) were extracted 2 d after plating on noncoated or collagen I–coated dishes. 30 or 60 μg of protein was blotted for N-cadherin, HA tag (to detect MKK7/JNK1), or tubulin. (B) BxPC3 cells expressing MKK7-JNK1 (a and b) or MKK7-KM-JNK1 (c and d) were cultured on noncoated (a and c) or collagen I–coated (b and d) dishes for 2 d. Bar, 100 μm. (C) 30 μg of protein from mock BxPC3 cells or cells expressing dominant-negative cJun (cJun-DN) were extracted 2 d after plating on noncoated or collagen I–coated dishes, resolved by SDS-PAGE, and blotted for N-cadherin, total cJun, or tubulin. (D) Mock BxPC3 cells (a and b) or cells expressing cJun-DN (c and d) were cultured on noncoated (a and c) or collagen I–coated (b and d) dishes for 2 d. Bar, 100 μm.
Mentions: Thus far, we have characterized a novel signaling pathway initiated by interactions with collagen I that requires both integrin and DDR1, is propagated through FAK and Pyk2, requires the p130CAS scaffold, and requires Rap1 GTPase. The outcome is activation of JNK1, using the upstream kinases MKK7 and MLK3. To confirm that activated JNK1 up-regulates expression of N-cadherin, we expressed constitutively active JNK1 in BxPC3 cells. It has been reported that fusing MKK7 to JNK1 results in constitutive JNK1 activity and activates c-Jun in the absence of any stimulus (Zheng et al., 1999). The JNK1 portion of the fusion protein is phosphorylated on both Thr183 and Tyr185 residues by the MKK7 portion of the fusion protein. Overexpression of MKK7-JNK1 increased N-cadherin expression even when cells were plated on noncoated dishes, whereas a control construct with kinase-dead MKK7 did not, showing that activation of JNK is sufficient to up-regulate N-cadherin (Fig. 8). Interestingly, overexpression of MKK7-JNK1 did not induce cell scattering on noncoated dishes, indicating that although JNK1 activation is necessary for cell scattering, it is not sufficient to induce cell scattering in the absence of a collagen I stimulus (Fig. 5 B). These data are consistent with our previous studies (Shintani et al., 2006a) and with data in Fig. S5 (available at http://www.jcb.org/cgi/content/full/jcb.200708137/DC1) showing that artificially up-regulating N-cadherin also did not induce cell scattering in the absence of a collagen I signal, indicating that up-regulation of N-cadherin is necessary, but not sufficient, for cell scattering.

Bottom Line: Focal adhesion kinase (FAK)-related protein tyrosine kinase (Pyk2) is downstream of DDR1, whereas FAK is downstream of alpha2beta1 integrin.Both receptor complexes rely on the p130 Crk-associated substrate scaffold.Interestingly, Rap1, but not Rho family guanosine triphosphatases, is required for the response to collagen I.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.

ABSTRACT
Tumor cells undergo epithelial-to-mesenchymal transition (EMT) to convert from a benign to a malignant phenotype. Our recent focus has been signaling pathways that promote EMT in response to collagen. We have shown that human pancreatic cancer cells respond to collagen by up-regulating N-cadherin, which promotes tumor growth, invasion, and metastasis. Initial characterization showed that knocking down c-Jun NH2-terminal kinase prevented N-cadherin up-regulation and limited tumor growth and invasion in a mouse model for pancreatic cancer. The current study was designed to understand the pathway from collagen to N-cadherin up-regulation. Initiation of the signal requires two collagen receptors, alpha2beta1 integrin and discoidin domain receptor (DDR) 1. Each receptor propagates signals through separate pathways that converge to up-regulate N-cadherin. Focal adhesion kinase (FAK)-related protein tyrosine kinase (Pyk2) is downstream of DDR1, whereas FAK is downstream of alpha2beta1 integrin. Both receptor complexes rely on the p130 Crk-associated substrate scaffold. Interestingly, Rap1, but not Rho family guanosine triphosphatases, is required for the response to collagen I.

Show MeSH
Related in: MedlinePlus