Proc. Natl. Acad. Sci. USA Vol. 95, pp. 9950–9955, August 1998 Developmental Biology Modulation of the chaperone heat shock cognate 70 by embryonic (pro)insulin correlates with prevention of apoptosis ENRIQUE J. DE LA ROSA*, ELENA VEGA-NU´NEZ˜ *, AIXA V. MORALES*†,JOSE´ SERNA,EVA RUBIO, AND FLORA DE PABLO‡ Department of Cell and Developmental Biology, Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientı´ficas, Vela´zquez144, E-28006 Madrid, Spain Communicated by William H. Daughaday, University of California, Irvine, CA, June 25, 1998 (received for review February 18, 1998) ABSTRACT Insights have emerged concerning insulin (13, 14), the physiological survival effect of (pro)insulin has function during development, from the finding that apoptosis received less attention. during chicken embryo neurulation is prevented by prepan- Molecular chaperones assist folding, translocation, and as- creatic (pro)insulin. While characterizing the molecules in- sembly of newly synthesized proteins (reviewed in refs. 15 and volved in this survival effect of insulin, we found insulin- 16) and also protect the cell from heat shock and other dependent regulation of the molecular chaperone heat shock physiological or environmental stresses, increasing cell sur- cognate 70 kDa (Hsc70), whose cloning in chicken is reported vival after stress (17, 18). The specific mechanisms involved in here. This chaperone, generally considered constitutively ex- the cell survival effect of some chaperones have nonetheless pressed, showed regulation of its mRNA and protein levels in been approached only recently (19–21). How extracellular unstressed embryos during early development. More impor- survival factors might modulate these chaperones and whether tant, Hsc70 levels were found to depend on endogenous these molecules mediate the factors’ antiapoptotic function (pro)insulin, as shown by using antisense oligodeoxynucleoti- have not been elucidated. des against (pro)insulin mRNA in cultured neurulating em- We have reported recently that a putative chicken heat bryos. Further, in the cultured embryos, apoptosis affected shock cognate (Hsc)70, a noninducible member of the heat mainly cells with the lowest level of Hsc70, as shown by shock 70-kDa protein family, is the antigen recognized by the simultaneous Hsc70 immunostaining and terminal de- monoclonal antibody PM1 (22, 23). The PM1-positive cells in oxynucleotidyltransferase-mediated UTP nick end labeling. the chicken embryonic retina in culture depend on exogenous These results argue in favor of Hsc70 involvement, modulated insulin and IGF-1 (24). Because insulin prevents apoptosis and by embryonic (pro)insulin, in the prevention of apoptosis maintains the PM1yHsc70-expressing cells, we have begun to during early development and suggest a role for a molecular explore a possible relationship between these phenomena that chaperone in normal embryogenesis. could prove to be part of the apoptosis prevention mechanism by embryonic (pro)insulin. We report here the isolation and Programmed cell death, a process recognized increasingly in characterization of the chicken hsc70 cDNA and its mRNA development (1), and the factors modulating survivalydeath of and protein expression from gastrulation to early organogen- embryonic cells are characterized poorly in the critical periods of esis. As hypothesized, endogenous (pro)insulin maintained gastrulation and neurulation. Apoptosis, as well as other devel- embryonic Hsc70 protein levels, and the apoptosis induced by opmental processes, is likely to be regulated by a complex, growth factor deprivation, which is attenuated by insulin, was changing combination of growth factors (2, 3). Some of these prevalent in cells with low Hsc70 levels. factors are well known for their actions in adult organisms, as is the case with the pancreatic hormone insulin. Preproinsulin EXPERIMENTAL PROCEDURES mRNA is also expressed in the early prepancreatic embryo, even Chicken Embryos and Treatments in Culture. before insulin-like growth factor I (IGF-I), at least in chicken (4, Fertilized 5) and Xenopus (6). The widespread distribution of preproinsulin White Leghorn eggs (Granja Rodrı´guez-Serrano, Salamanca, mRNA during gastrulation and neurulation, as well as the similar Spain) were incubated at 38.4°C, 60–90% relative humidity. The embryos were staged according to Hamburger and Ham- broad distribution of the insulin receptor mRNA (5) and protein ilton (25). Embryos at 1.5 days of development (E1.5) were (7), potentially implicates (pro)insulin in the regulation of cellular cultured in chemically defined medium as described (4, 26). events in early development. Indeed, we demonstrated previously Where indicated, embryos were treated with purified chicken that insulin is an autocrineyparacrine survival signal (5). Inter- insulin (Litron Lab, Rochester, NY), recombinant human ference with embryonic (pro)insulin and insulin receptor expres- insulin (a kind gift from Eli Lilly), or 12.5 nmol of the indicated sion using antisense oligodeoxynucleotides (ODNs) thus in- ODN as described (see refs. 5 and 26 for extensive details and creased apoptosis, both in culture and in ovo (5), supporting the controls). All ODNs were synthesized as phosphorothioate involvement of insulin signaling in the regulation of programmed derivatives by Oligos Etc. (Wilsonville, OR). Sequences were cell death. In addition, exogenous insulin suppresses apoptosis of ASA, 59-GAGAGCCATGATGAG-39; ASB, 59-GCTCGA- chicken embryo lens cells (8) and maintains the survival of CATCCCGTC-39; RAN, 59-TAACGGTAACGGAGG-39; migratory neural crest precursors (9), among other effects on neural cells (reviewed in ref. 10). Although another member of this family, IGF-I, is widely recognized as a survival factor for Abbreviations: Hsc70, heat shock cognate, 70 kDa; IGF, insulin-like many cell types (10–12), and its possible mechanism of action growth factor; ODN, oligodeoxynucleotide; En, embryonic day n; y TUNEL, terminal deoxynucleotidyltransferase-mediated UTP nick through the pI3 kinase Akt pathway is beginning to be elucidated end labeling. Data deposition: The sequences reported in this paper have been The publication costs of this article were defrayed in part by page charge deposited in the GenBank database (accession no. AJ004940). *E.J.d.l.R., E.V.-N., and A.V.M. contributed equally to this work. payment. This article must therefore be hereby marked ‘‘advertisement’’ in †Present address: Developmental Genetics Laboratory, ICRF, Lon- accordance with 18 U.S.C. §1734 solely to indicate this fact. don, UK. © 1998 by The National Academy of Sciences 0027-8424y98y959950-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: cibfp1f@ PNAS is available online at www.pnas.org. fresno.csic.es. 9950 Downloaded by guest on October 1, 2021 Developmental Biology: de la Rosa et al. Proc. Natl. Acad. Sci. USA 95 (1998) 9951 SS, 59-CTCATCATGGCTCTC-39; ASIGF-I 59-TTTTTC- ma) in glycerol:PBS (9:1). Pyknotic nuclei were observed CATTGCTTC-39). The inhibitors Ac-YVAD-CHO (for under epifluorescence illumination in a Zeiss Axioscop. caspase 1) and Ac-DEVD-CHO (for caspase 3) or a control Terminal Deoxynucleotidyltransferase-Mediated UTP Nick peptide inhibitor, Ac-LLR-CHO (leupeptin), were also used End Labeling (TUNEL) and Hsc70 Staining and Flow Cytom- (Bachem). The inhibitors were added to the agarose under the etry. Cultured embryos were processed as above. Aliquots of embryo and to the medium at the indicated concentrations. the cell suspension, typically 50,000 cells, were stained with rat After 5 h, a second dose was added to the medium. mAb anti-Hsc70 (1.7 mgyml) or with Igs as negative control, Reverse Transcription PCR Cloning, cDNA Library Screen- followed by biotinylated goat anti-mouse Ig (1y200 dilution, ing and Sequence Analysis. Total RNA was extracted from the Amersham) and then with Cy2-streptavidin (1y200 dilution, brain of embryos at 4.5 days of development (E4.5) by the Amersham). Fragmented DNA was stained by the TUNEL RNAzol method (Biotecx Laboratories Inc., Houston). The method in the same cell suspensions and was permeated with RNA was reverse-transcribed using the First-Strand cDNA 0.6% (wtyvol) Triton X-100 in 23 SSPE. Samples were Synthesis Kit (Pharmacia), with the antisense primer 59- incubated1hat37°C with 50 ml of a solution containing GGIGGIGAIGA(CyT)TT(CyT)GA(CyT)AA-39. The re- biotin-16-dUTP and terminal-deoxynucleotidyl-transferase, as verse transcription product was subjected to PCR amplifica- indicated by the manufacturer (Boehringer Mannheim). The tion using 10 mM Tris, 50 mM KCl, 200 mM each dNTP, 1 mM reaction was terminated by a 2-h incubation in 23 SSPE at MgCl2, 0.5 nM antisense primer, and 0.5 nM sense primer 65°C. The end-labeling with biotin-16-dUTP was visualized 59-CCICCIACIA(AyG)IACIAT(AyG)TC(AyG)TG-39; with Cy3-streptavidin (1y100, Amersham). Flow cytometry primers were derived from the known peptides sequence (23). data collection and multiparameter analysis were done in an The 309-bp amplified product was sequenced automatically by EPICS XL Coulter Cytometer, as described (28). the dideoxynucleotide method (Perkin–Elmer) and cloned into a pGEM-T (Promega) vector. The sequence was com- RESULTS pared by using the GCG software package and showed the highest identity with the human hsc70 gene. This fragment was Cloning of Chicken hsc70 cDNA. The biochemical data labeled with digoxigenin-dUTP (Boehringer Mannheim) by indicating that the antigen recognized by mAb