Reactivation of developmental programs: the cAMP-response element-binding protein pathway is involved in hydra head regeneration

Abstract

Hydra regenerate throughout their life. We previously described early modulations in cAMP-response element-binding protein (CREB) DNA-binding activity during regeneration. We now show that the Ser-67 residue located in the P-box is a target for post-translational regulation. The antihydra CREB antiserum detected CREB-positive nuclei distributed in endoderm and ectoderm, whereas the phosphoSer133-CREB antibody detected phospho-CREB-positive nuclei exclusively in endodermal cells. During early regeneration, we observed a dramatic increase in the number of phospho-CREB-positive nuclei in head-regenerating tips, exceeding 80% of the endodermal cells. We identified among CREB-binding kinases the p80 kinase, which showed an enhanced activity and a hyperphosphorylated status during head but not foot regeneration. According to biochemical and immunological evidence, this p80 kinase belongs to the Ribosomal protein S6 kinase family. Exposure to the U0126 mitogen-activated protein kinase kinase inhibitor inhibited head but not foot regeneration, abolished CREB phosphorylation and activation of the early gene HyBra1 in head-regenerating tips. These data support a role for the mitogen-activated protein kinase/ribosomal protein S6 kinase/CREB pathway in hydra head organizer activity.

Fig. 1.

The hydra CREB protein is a phosphoprotein. (A) Map of the H. viridissima, Hv, and 6HisCREB proteins. The gray boxed sites indicate phosphorylation sites also present in vertebrate CREB/CREM proteins. P, P-box; BD, binding domain; LZ, leucine zipper domain; Cv, H. viridissima. (B) Alignment of the human (Hs), mouse (Mm), songbird (Pg), Drosophila (Dm), and hydra (H. viridissima, Hv, and S67->A67 mut) P-box sequences to the phosphopeptide used to raise the phosphoSer133-CREB antibody (15). (C) In vitro phosphorylation of 6HisCREB S67 (lanes 1–4) and 6HisCREB A67 (lanes 5–8) proteins bound to Ni-agarose beads, treated with PKA in the presence of [γ-³²P]ATP, and detected by autoradiography (Top) and Western analysis by using the anti-P-CREB antibody (Middle). The amount of substrate is shown (Bottom) (hyCREB24 antibody). PKI, PKA inhibitor. (D) Hv WCE contain CREB kinase activities. (Left) WCE immunoprecipitated with hyCREB24 and tested in a kinase assay. IS, immune antiserum; PIS, preimmune serum. The arrow indicates a 32-kDa phosphorylated product (lane 2). (Right) Hv WCE purified on 6HisCREB protein submitted to SPKA. Phosphorylated products were detected by autoradiography. C30, C20: full-length and prematurely terminated 6HisCREBfusion proteins.

Fig. 2.

Immunodetection of CREB- and P-CREB-expressing cells in hydra (Hv). The hyCREB24 (A, E, and H) and anti-P-CREB (B–D, F, G, I, J) antibodies were used in intact hydra (A–D), head-(E–G), and foot-(H–J) regenerating halves 4 h after midgastric section. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). Arrowheads: ectodermal positive nuclei; arrows: endodermal positive nuclei; end, endodermal cells; ect, ectodermal cells; hyp, hypostome; t, tentacles; brackets, stump area. (D, G, and J) P-CREB-positive nuclei in confocal views. (Bars = 100 μm in A–C, 40 μm in E, F, H, and I.)

Fig. 3.

Temporal regulation of CREB-binding kinases during regeneration. (A) CREB-binding proteins were purified from WCE prepared at indicated time points from either head- (lanes 3, 5, 7, 9, 13, 15, 17, and 19) or foot- (lanes 2, 4, 6, 8, 12, 14, 16, and 18) regenerating halves and subsequently tested in GKA. Ho, H. oligactis. (B) The extracts used in A were assayed in SPKA to evaluate the modulations in p80 and CREB (C30) phosphorylation levels. (C) GKA showing similar levels in CREB-binding kinase activities in WCE prepared from nonregenerating halves obtained from frozen hydra.

Fig. 4.

The hydra factors released on bisection modulate regeneration kinetics and p80 kinase activity. (A) Head regeneration in hydra (Hv) bisected in different volumes and left in the same medium for 3 days. We scored the emergence of tentacle rudiments at indicated time points. Triplicates of 30 hydra were tested in each condition. (B) Kinase assay (SPKA) testing Hv CREB-binding proteins purified from 40 hydra allowed to regenerate for 1 h in indicated volumes. (Upper) The p80 (arrow) and CREB (C30) display parallel phosphorylation variations according to the hydra density. (Lower) Control Western analysis [anti-actin, Abcam (Cambridge, U.K.), 1/5,000].

Fig. 5.

Biochemical and immunological characterization of the p80 CREB kinase. (A) GKA showing similar levels in p80 kinase activity in the presence (+, lane 2) or absence (–, lane 3) of substrate in the gel. M, marker. (B) SPKA showing the p80 and 6HisCREB phosphorylation levels when CREB-binding proteins were treated with inhibitors for PKC (Bim 3 μM, lanes 2, 6, 7, and 11), PKA (PKA inhibitor, 9 units, Biolabs, lane 3), CaMKII (KN-62 4 μM, lanes 9–11), and/or competitors for PKC/RSK (Ser-25 peptide 19–31, 0.42 mM, lanes 5, 6), extracellular response kinase (ERK)/MAP kinases (APRTPGRR, 0.69 mM, lane 8), and CaMKII (autocamtide-2 0.44 mM, lane 10), 63×, 100×, and 64× in excess to 6HisCREB, respectively. Lanes 1 and 4, untreated controls for lanes 2, 3, and 5–11, respectively. (C) Coimmunoprecipitation of the p80 kinase from Hv (lanes 1, 2, 5, and 6) and H. viridissima (lanes 3, 4, 7, and 8). WCE or NE immunoprecipitated with the anti-hyCREB24 antibody(ies) were analyzed with the biotinylated-panRSK antibody in Western blot. A specific p80 band was detected (lanes 1, 3, 5, and 7, arrow). Pis, preimmune serum; C, A431 cell lysate positive control extract.

Fig. 6.

Dose-dependent inhibition of head but not foot regeneration on exposure to the mitogen-activated protein kinase kinase inhibitor U0126. (A) For head regeneration, appearance of tentacle rudiments was recorded on lower halves. (B) For foot regeneration, peroxidase staining (36) was performed on upper halves after 3 days. Forty animals were taken for each condition. (C–F) Immunodetection of either CREB- (C) or P-CREB- (D–F) positive nuclei in regenerating stumps 4 h after bisection exposed to U0126 (20 μM) or DMSO (E Lower)4′,6-diamidino-2-phenylindole (DAPI) staining. (C–E) Head-regenerating tips. (F) Foot-regenerating tip. Arrows, endodermal positive nuclei; arrowheads, ectodermal positive nuclei. (G–I) HyBra1 whole-mount in situ hybridization performed on 10-h-regenerating upper (Left) and lower halves exposed or not to U0126. Arrows and arrowheads show adult heads and head-regenerating tips, respectively.