Modulation of the chaperone heat shock cognate 70 by embryonic (pro)insulin correlates with prevention of apoptosis

Abstract

Insights have emerged concerning insulin function during development, from the finding that apoptosis during chicken embryo neurulation is prevented by prepancreatic (pro)insulin. While characterizing the molecules involved in this survival effect of insulin, we found insulin-dependent regulation of the molecular chaperone heat shock cognate 70 kDa (Hsc70), whose cloning in chicken is reported here. This chaperone, generally considered constitutively expressed, showed regulation of its mRNA and protein levels in unstressed embryos during early development. More important, Hsc70 levels were found to depend on endogenous (pro)insulin, as shown by using antisense oligodeoxynucleotides against (pro)insulin mRNA in cultured neurulating embryos. Further, in the cultured embryos, apoptosis affected mainly cells with the lowest level of Hsc70, as shown by simultaneous Hsc70 immunostaining and terminal deoxynucleotidyltransferase-mediated UTP nick end labeling. These results argue in favor of Hsc70 involvement, modulated by embryonic (pro)insulin, in the prevention of apoptosis during early development and suggest a role for a molecular chaperone in normal embryogenesis.

Figure 1

Predicted amino acid sequence of chicken Hsc70 (cHSC70), compared with chicken Hsp70 (cHSP70), Grp78 (cGRP78), and human Hsc70 (hHSC70). The amino acids conserved in all sequences are in black boxes, those conserved only in some of the sequences are in gray boxes. European Molecular Biology Laboratory nucleotide sequence database accession number for chicken Hsc70 is AJ004940.

Figure 2

Expression of Hsc70 mRNA and protein in early embryos. (A) Northern blot analysis of hsc70 mRNA in whole chicken embryos at the indicated ages. A single 2.2-kb transcript was revealed. Methylene blue staining of the 18S rRNA in the same blot (Lower) allowed for loading correction. The relative densitometric values (mean of three blots) after 18S correction were: 1 (E0.5), 1 (E1), 2 (E1.5), and 2.8 (E2.5). (B) Embryonic extracts of the indicated ages were subjected to SDS-PAGE and immunoblotting. Staining with antiserum anti-Hsc70^pep revealed a 73-kDa band at all ages. In a parallel blot stained with mAb anti-Hsp70, only a faint band was observed at E0.5, despite the fact that the blot was exposed 8-fold longer than that stained for Hsc70. All the blots were restained for tubulin to allow for loading correction. The relative Hsc70 densitometric values (mean of four blots) after tubulin correction were: 1 (E0.5), 1 (E1), 1.3 (E1.5), and 1.8 (E2.5).

Figure 3

Protection from apoptosis by caspase inhibitors and insulin. E1.5 embryos were cultured in basal medium alone (Med) or with the indicated concentrations of leupeptin (Leup), caspase 1 and 3 inhibitors (C-Inh), chicken insulin (Ins), or 100 nM insulin and 0.5 mM each caspase inhibitors together. After 10 h of culture, apoptosis was quantified in dissociated cells. Six hundred cells were counted per experimental point. The values represent the mean ± SEM of three to five cultured embryos in each of two to eight different experiments and are expressed relative to the embryos cultured in medium alone. ∗, P < 0.001 with respect to the medium alone.

Figure 4

Exogenous insulin increases Hsc70 mRNA and protein levels in cultured embryos. (A) Northern blot analysis of hsc70 mRNA in whole chicken embryos cultured in basal medium (M) or in the presence of insulin (Ins) for 12 h. The single 2.2-kb transcript was quantified relative to methylene blue-stained 18S rRNA (Lower). The relative densitometric values (mean of two blots) were 1 (M), 1.3 (10⁻⁸ M insulin) and 1.2 (10⁻⁷ M insulin). (B) Extracts of embryos cultured in basal medium or in the presence of insulin for 12 h were subjected to SDS-PAGE and immunoblotting. The staining with antiserum anti-Hsc70^pep revealed a 73-kDa band that was quantified relative to tubulin (Lower). The relative Hsc70 densitometric values (mean of four blots) were 1 (M), 1.16 (10⁻⁹ M insulin), 1.25 (10⁻⁸ insulin).

Figure 5

Hsc70 levels decrease after treatment with (pro)insulin antisense oligonucleotides. (A) Two representative blots, illustrating the different treatments, with extracts from cultured E1.5 embryos treated with the indicated insulin antisense (ASA, ASB), IGF-I antisense (ASIGF-I), or control (RAN, SS) ODNs, and 10⁻⁷ M chicken insulin where indicated. After 10 h in culture, the extracts from individual embryos were subjected to SDS-PAGE and immunoblotting with mAb PM1 or with antiserum anti-Hsc70^pep. Blots were restained for tubulin for loading correction and quantified by densitometry. (B) Quantitation of ODN effect on Hsc70 levels. Values represent the mean ± SEM of two to three replicate immunoblots from each of two to three embryos treated per point in each of two to three independent experiments and are expressed relative to control RAN ODN treated embryos in the same experiment. ∗, P < 0.025 relative to the RAN treatment; #, P < 0.025, relative to the ASB treatment.

Figure 6

Flow cytometric analysis of Hsc70- and TUNEL-stained cells from E1.5 embryos cultured for 6 h in basal medium. Double TUNEL and Hsc70 immunostaining was performed in dissociated cells. (A) One representative Hsc70 staining (continuous line) of total cells (of 15 embryos in five independent experiments). Nonimmune Ig was used as a control (dotted line). (B) Hsc70 immunofluorescence levels were also analyzed in the TUNEL-stained cells. Note that 45% of apoptotic cells coincided with the area considered negative for Hsc70 (dotted line) and 90% of the TUNEL-stained cells showed an Hsc70 immunofluorescence signal below the mean signal of the total cell population. The profiles were similar for embryos cultured with insulin, but the proportion of apoptotic cells was lower.