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First published online 29 May 2007
doi: 10.1242/jcs.006247

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Non-classical export of epimorphin and its adhesion to v-integrin in regulation of epithelial morphogenesis

¹ Department of Morphoregulation, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
² Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 977, Berkeley, CA 94720, USA
³ Mayo Clinic Cancer Center, 4500 San Pablo Road, Jacksonville, FL 32224, USA
⁴ Mayo Clinic Proteomics Research Center, 200 First St. SW, Rochester, MN 55905, USA

View larger version (45K): [in this window] [in a new window]   Fig. 1. Extracellular secretion of epimorphin in mammary stromal cells. (A) Epimorphin is produced in the E-cadherin-negative compartment in the mammary gland. Cryosections (left panel) and primary cells (right panel) of glands from mid-pregnant (day 13) mice (ICR, Shimizu Laboratory Supplies) were stained with goat anti-E-cadherin antibodies (E-cadherin; green) and affinity-purified rat anti-epimorphin antibodies (Epm; red). Non-immunized rat serum and secondary antibodies did not label cells. Bars, 100 µm. (B) Secretion of the 30 kDa soluble epimorphin but not of highly similar syntaxin 1A. Epimorphin-producing mammary cell populations were transfected with expression plasmid for T7-tagged epimorphin or syntaxin 1A (TE and TS, respectively), and proteins were immunoprecipitated from the whole-cell culture (cell + sup), cell (cell) or supernatant (sup) with pan-syntaxin antibodies followed by western blot analysis using anti-T7 mAb. Loaded amount of supernatant was five times higher than that of cell lysate.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Extracellular secretion of epimorphin is dependent on the SNARE/TM domain. (A) Schematic of chimeric and mutant constructs. 3-Hlx, triple-helical N-terminal domain; SNARE, SNARE domain; TM, transmembrane domain. Epimorphin (Epm) is shown in red, syntaxin 1A is shown in blue. (B) Cell surface presentation of epimorphin (TE), syntaxin 1A (TS) and the domain-swapped molecules (TES, TSE), but not Epm lacking the SNARE/TM domain (TES) or the cytoplasmic β-actin protein. PT67 cells transiently transfected with indicated constructs were labeled with membrane-impermeant biotinylation reagents. Cell lysates were incubated with streptavidin-agarose beads and bound (Ex) or unbound (In) fractions were probed with antibodies against β-actin and the T7 tag. (C) Transfection with TE or TSE causes secretion of a soluble form of the molecules into the medium (sup), whereas transfection with TS, TES, or TES does not. Loaded amount of supernatant was five times higher than that of cell lysate. (D) Proteomic analysis of secreted 30 kDa epimorphin. Supernatant of TE transfectant was collected, the 30 kDa soluble epimorphin was retrieved with anti-T7-coated beads and separated by SDS-PAGE. Protein amount from cells of one culture dish (10-cm diameter) was visible with Coomassie Brilliant Blue staining. The protein band was isolated and tryptic fragments were analyzed by mass spectrometry (MS-MS). Identified sequences are underlined. The most C-terminal peptide identified ends with glutamic acid (E), indicating the cleavage site.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Identification of critical amino acids for epimorphin secretion. (A) Comparison of the sequences close to the cleavage site of epimorphin (Epm) and conserved syntaxin family members. Syntaxin 1A, syn1; syntaxin 3 (NM001025307), Syn3; and syntaxin 4 (NP033320), Syn4. Histidine H246 (red) is conserved in epimorphin from mouse (m, D10475), human (h, D14582), quail (q, AB076670) and sheep (s, E32546); other syntaxins have arginine R246 (blue). Adjacent to H246, the KEE sequence (red-yellow-red) is also conserved in epimorphin, whereas the VXD sequence (blue-black-blue) occupies this position in syntaxin 1A and syntaxin 3. (B) H246 is crucial for processing and secretion of epimorphin. The H246R mutation (CATAGG) in the T7-epimorphin sequence dramatically suppressed the secretion of 30 kDa epimorphin. The R246H (AGGCAT) mutation in T7-syntaxin-1A resulted in the secretion of a 30 kDa form.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Induction of epimorphin secretion in stable transfectants. (A) Appearance of a 34 kDa full-length epimorphin at cell surface (Ex) and a 30 kDa form in the supernatant (Sup) of PT67 cells stably expressing the TE transgene following treatment with subtoxic levels of cycloheximide (CHX), actinomycin D (Acti-D), camptothecin (Camp), or the Ca2+ ionophore A23187. Treatment with DMSO (–) or the Ca2+-chelator EDTA did not lead to membrane translocation. (B) Epimorphin secretion is not a consequence of cell death. The calpain inhibitor Calpeptin decreased epimorphin secretion by partially inhibiting cycloheximide-dependent cell death. However, neither cell stresses (0.5 mM EDTA) nor temporal heat shock (42°C for 1.5 hours at day 2) resulted in a detectable increase in epimorphin secretion. Cell viability at day 3 was quantified with alamarBlueTM assay with non-treated cells being treated as 100% viable. Data are the mean ± s.d., n=4, *P<0.05. (C) Epimorphin coprecipitates with annexin II and synaptotagmin1 (full-length 65 kDa synaptotagmin and the 40 kDa extravesicular domain) from lysate of the transfectants, regardless of cycloheximide treatment (upper panel). Epimorphin is secreted to medium in response to cycloheximide-treatment in a secretory complex with annexin II and the extravesicular domain of synaptotagmin, both of which directly bind to phosphatidylserine (middle panel). Cycloheximide induces phosphatidylserine externalization as judged by binding of FITC-labeled annexinV (Biovision) to the non-permeabilized cells (lower panel). Cell nuclei were visualized with propidium iodide (PI) after permeabilization. Bar, 25 µm. (D) Anti-epimorphin antibodies (red) bind to the surface of non-permeabilized PT67 cells expressing TE transgene following treatment with CHX, whereas binding of anti-β-actin antibodies (green) requires cell permeabilization. Bar, 10 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Cells adhere to secreted epimorphin and activate focal adhesion kinase. (A) SCp2 cells adhere to intact secretory protein complex containing 30 kDa soluble epimorphin, 40 kDa synaptotagmin and annexin II, accompanied by FAK activation. This activity is selectively blocked by anti-epimorphin antibodies. The immobilization procedure was carried out under physiological conditions using the T7-tag as the target site for the immobilization. Western blotting of the immobilized materials confirmed successful immobilization of 30 kDa epimorphin, annexin II and 40 kDa synaptotagmin after incubation with supernatant from TE transfectants but not from the control cells (Vector). The amount of captured epimorphin on the substrate was about 100 ng/cm2, as determined by a luminescent image analyzer Las1000plus using purified recombinant epimorphin as reference. The number of cells bound to each well in 4 hours was counted and the relative number to collagen-coated wells was calculated. Data are the mean ± s.d., n=4, *P<0.05. P-FAK, FAK phosphorylated at Y397. Bar, 50 µm. (B) Recombinant forms of secreted epimorphin r-Epm30 (aa 1-245) and r-Epm (aa 1-188), but not recombinant epimorphin SNARE domain r-SNARE (aa 189-265), bind to SCp2 cells with FAK/ERK activation. Images show cells starved for 24 hours, incubated with the indicated recombinant protein for 30 minutes; bound protein (red) and E-cadherin (green). Bars, 20 µm. Blots on the right show phosphorylation of FAK in cells treated with a recombinant protein for 15 and 30 minutes (first column of blots). phosphorylation of ERK but not EGF receptor was detected in cells treated with r-Epm for 30 minutes (second column of blots).

View larger version (34K): [in this window] [in a new window]   Fig. 6. Cell adhesion to epimorphin is mediated by v-integrins. (A) Quantification of HMEC adhesion to recombinant epimorphin in the presence of anti-integrin antibodies. Adhesion to r-Epm is inhibited by antibodies that block v- and β1-integrins (left). In the presence of function-stimulating antibody for β1-integrin (st) most of the cells bound to epimorphin within 2 hours (right). v; clone P3G8, v'; clone AV1. Data are the mean ± s.d., n=6, *P<0.05 vs r-Epm only. (B) Epimorphin adhesion-induced FAK phosphorylation is inhibited by antibodies against v- and β1-integrins. (C) Immobilized epimorphin specifically binds to v- and β1-integrins from HMECs lysates. Epimorphin beads selectively pull down 120-kDa to 140-kDa proteins (non-reduced condition) from HMEC-surface components labeled with membrane-impermeant biotinylation reagents. HRP-labeled streptavidin was used to visualize the proteins (left blot). Immunoblot of the proteins bound to r-Epm beads under the reduced condition. The cell surface proteins that bound to Epm beads appeared to include v- and β1-integrins. FN, fibronectin (right blot). (D) Function-blocking antibodies against v- and β1-integrins block adhesion of HSC-5 and HUVECs, whereas inhibition of v- and vβ5 integrins blocks adhesion of A549 and MCF7 cells. Data are the mean ± s.d., n=6, *P<0.05 vs control. (E) The transient silencing of v-integrin results in a dramatic attenuation of epimorphin association of HMECs. The cells infected with lentivirus without (NT) or with the knockdown construct HE4 or HE5 were assessed for the expression of v- and β1-integrins and cellular adhesion to r-Epm at day 3. Data are the mean ± s.d., n=4, *P<0.05 vs NT. (F) Epimorphin beads pull down purified vβ5-integrin but not 1β1-integrin, and this integrin-epimorphin association is not affected by addition of RGDS or RGES peptide. v- or 1β1-integrin bound to the beads was detected with antibodies against v-integrin or β1-integrin, respectively.

View larger version (41K): [in this window] [in a new window]   Fig. 7. Inhibition of v-integrins blocks epimorphin-mediated epithelial morphogenesis. (A) Extracellular epimorphin stimulates luminal or branching morphogenesis in HMECs, depending upon mode of presentation. In suspension culture HMECs form clearly rounded spherical aggregates after 24 hours (see top two micrographs). Adenovirus carrying signal peptide-tagged epimorphin (Av-SE) was added to the suspension culture for the final two hours (cells form well-packed aggregate and Av-SE infects only outer cells in the aggregate) for polar presentation, or first 2 hours (when cells remain single and Av-SE infects all cells in the aggregate) for apolar presentation; embedding of the resultant cell aggregates in 3D collagen gels produced branching or luminal morphogenesis, respectively. Bar, 50 µm. Blot on the right shows HMECs treated with Av-SE producing 37 kDa glycosylated epimorphin. (B) Antibodies against v-integrin block both branching and luminal morphogenesis. Data are the mean ± s.d., n=3. The phenotypic appearance (branching or luminal) was evaluated as described previously (Hirai et al., 1998).

View larger version (59K): [in this window] [in a new window]   Fig. 8. Cell surface presentation and secretion of epimorphin and its extracellular action for epithelial morphogenesis. Intracellular epimorphin becomes complexed with synaptotagmin (syt) and annexin II, both of which can directly bind to phosphatidylserine at the inner leaflet of the plasma membrane. In response to stress and/or Ca2+ influx, this release complex translocates across the membrane with externalization of phosphatidylserine. The extracellularly presented epimorphin is then cleaved at the SNARE domain and the secreted form of epimorphin is captured by v-integrin on the target epithelia leading to FAK activation and initiation of morphogenesis. The role of cytoplasmic epimorphin as a t-SNARE protein in SNARE-mediated membrane fusion (inset). EX, extracellular space. IN, intracellular space. Syb, synaptobrevin, Epm, epimorphin.

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