STEPWISE EVOLUTION OF RESISTANCE TO TOXIC CARDENOLIDES VIA GENETIC SUBSTITUTIONS IN THE NA+/K+‐ATPASE OF MILKWEED BUTTERFLIES (LEPIDOPTERA: DANAINI)

Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to cardenolides, because their target site, the sodium pump (Na⁺/K⁺‐ATPase), has evolved amino acid substitutions that reduce cardenolide binding (so‐called target site insensitivity, TSI). Because many, but not all, species of milkweed butterflies (Danaini) are associated with cardenolide‐containing host plants, we analyzed 16 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na⁺/K⁺‐ATPase gene and by enzymatic assays with extracted Na⁺/K⁺‐ATPase. Here we report that sensitivity to cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic cardenolides. Thus, the interplay between bottom‐up selection by plant compounds and top‐down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins.     

Chromatin compaction and cell death by high molecular weight FGF-2 depend on its nuclear localization, intracrine ERK activation, and engagement of mitochondria

Fibroblast growth factor 2 (FGF-2) is produced as CUG-initiated, 22-34 kDa or AUG-initiated 18 kDa isoforms (hi- and lo-FGF-2, respectively), with potentially distinct functions. We report that expression of hi-FGF-2 in HEK293 cells elicited chromatin compaction preceding cell death with apoptotic features. Nuclear localization of the intact protein was required as expression of a non-nuclear hi-FGF-2 mutant failed to elicit chromatin compaction. Equally ineffective, despite nuclear localization, was the over-expression of the 18 kDa core sequence (lo-FGF-2). Chromatin compaction by hi-FGF-2 was accompanied by increased cytosolic cytochrome C, and was attenuated either by over-expression of Bcl-2 or by a peptide inhibitor of the pro-apoptotic protein Bax. In addition hi-FGF-2 elicited sustained activation of total and nuclear extracellular signal regulated kinase (ERK1/2) by an intracrine route, as it was not prevented by neutralizing anti-FGF-2 antibodies. Inhibition of the ERK1/2 activating pathway by dominant negative upstream activating kinase, or by PD 98059, prevented chromatin compaction by hi-FGF-2. ERK1/2 activation was not affected by the Bax-inhibiting peptide suggesting that it occurred upstream of mitochondrial involvement. We conclude that the hi-FGF-2-induced chromatin compaction and cell death requires its nuclear localization, intracrine ERK1/2 activation and mitochondrial engagement.     

Administration of FGF-2 to the Heart Stimulates Connexin-43 Phosphorylation at Protein Kinase C Target Sites

Fibroblast growth factor-2 (FGF-2) confers acute, preconditioning-like cardiac resistance to ischemic injury in a protein kinase C (PKC)-dependent fashion. One of the downstream targets of PKC is the gap junction protein connexin-43 (Cx43). We thus examined the effects of FGF-2 on Cx43 phosphorylation at specific PKC sites in the adult heart. Rat hearts perfused ex vivo for 20 min with an FGF-2-containing solution displayed increased levels of phosphorylated 44-45 kDa Cx43, assessed by western blotting. In addition, FGF-2 significantly upregulated phosphorylation of the PKC target serines 262 and 368 on Cx43 at intercalated disks, assessed using phosphospecific antibodies in immunolocalization and western blotting assays. Our data show that FGF-2, administered by perfusion, can alter the phosphorylation status of Cx43 at cardiomyocyte intercalated disks, and suggest a link between phosphorylation of Cx43 at specific PKC sites and FGF-2 cardioprotection.     

The subcellular distribution of protein kinase Calpha, -epsilon, and -zeta isoforms during cardiac cell differentiation.

There is little information on the molecular events that control the subcellular distribution of protein kinase C during cardiac cell differentiation. We examined protein kinase C activity and the subcellular distribution of representatives of the "classical," "novel," and "atypical" protein kinase C's in P19 murine teratoma cells induced to undergo differentiation into cardiac myocytes by the addition of dimethylsulfoxide to the medium (Grepin et al., Development 124, 2387-2395, 1997). Differentiation was assessed by the presence of striated myosin, a morphological marker for cardiac cells. Addition of dimethyl sulfoxide to the medium resulted in the appearance of striated myosin by 10 days postincubation. Immunolocalization and Western blot studies revealed that a significant proportion of protein kinase Calpha, -epsilon, and -zeta were associated with the particulate fraction in P19 cells prior to differentiation. Differentiation into cardiac cells resulted in a translocation of protein kinase C activity from the particulate fraction to cytosol and localization of most of protein kinase Calpha, -epsilon, and -zeta to the cytoplasmic compartment. The total cellular protein kinase C activity was unaltered during differentiation. The translocation of protein kinase C activity during differentiation of P19 cells into cardiac myocytes was associated with a decrease in the levels of cellular 1, 2-diacyl-sn-glycerol. The cellular levels of phosphatidylserine and phosphatidylinositol did not change during differentiation. Addition of 1,2-dioctanoyl-sn-glycerol, a cell-permeant 1, 2-diacyl-sn-glycerol analog, reversed the differentiation-induced switch in the relative distribution of protein kinase C activity and dramatically increased the association of protein kinase Calpha with the particulate fraction. Addition of 1,2-dioctanoyl-sn-glycerol did not reverse the pattern of distribution for protein kinase Cepsilon or -zeta. The results indicate that protein kinase C activity and protein kinase Calpha, -epsilon and -zeta isoforms are redistributed from the particulate to the cytosolic fraction during differentiation of P19 cells into cardiomyocytes. The mechanism for the redistribution of protein kinase Calpha may be related to the reduction in the cellular 1,2-diacyl-sn-glycerol levels that accompany differentiation.     

Calcium protects pituitary basic fibroblast growth factors from limited proteolysis by co-purifying proteases

Extracts from bovine pituitaries and other tissues contained basic fibroblast growth factor-like peptides of 22-26 kda, co-fractionating with smaller, 16-20 kda bFGFs. Heparin-bound, 22-26 kda bFGFs were converted to smaller, heparin-binding forms by tryptic proteolysis. In solution, 22-26 kda bFGFs were converted to smaller, heparin-binding forms by an activity present in pituitary extracts. Calcium protected higher molecular weight pituitary bFGFs from truncation by the endogenous activity, which was not acid-activated, co-purified with bFGF during heparin-sepharose chromatography, remained operant at high salt concentrations and was inhibited by phenylmethan-sulfonyl fluoride.     

Cell-cycle dependent anti-FGF-2 staining of chicken cardiac myocytes: Movement from chromosomal to cleavage furrow- and midbody-associated sites

Fibroblast growth factor-2 (FGF-2) promotes cardiac myocyte proliferation and has been detected in extracellular as well as cytoplasmic and nuclear compartments. As a first step in examining the participation of intracellular FGF-2 in cardiac myocyte cell cycle we have investigated its localization in proliferative chicken cells during interphase and the various stages of mitosis in culture. We have used a previously characterized and affinity-purified anti-FGF-2 antibody preparation which recognizes the 19-22 kDa variants of chick FGF-2. By immunofluorescence, bright, punctate anti-FGF-2 labelling was observed in 26% of interphase nuclei from myocytes derived from 5 day embryonic heart ventricles; these nuclei were positive for anti-bromodeoxyuridine staining indicating that they are at the S- or G2 phase of the cell cycle. In prophase and metaphase, bright anti-FGF-2 staining was detected in apparent association with chromosomes. During anaphase, however, anti-FGF-2 staining dissociated from chromosomal locations distinctly remaining in strand-like structures in the area of ensuing cleavage furrow formation. In late telophase and cytokinesis, strong staining persisted in the area of the midbody and reappeared in a small fraction of newly formed daughter nuclei. Absorption of the antibody preparation with immobilized FGF-2 eliminated all staining. This dynamic pattern of anti-FGF-2 staining suggests that chick FGF-2 or immunologically related protein(s) not only increase in DNA-synthesizing nuclei but they may play a role in subsequent stages of mitosis and cytokinesis.     

Administration of FGF-2 to the heart stimulates connexin-43 phosphorylation at protein kinase C target sites

Fibroblast growth factor-2 (FGF-2) confers acute, preconditioning-like cardiac resistance to ischemic injury in a protein kinase C (PKC)-dependent fashion. One of the downstream targets of PKC is the gap junction protein connexin-43 (Cx43). We thus examined the effects of FGF-2 on Cx43 phosphorylation at specific PKC sites in the adult heart. Rat hearts perfused ex vivo for 20 min with an FGF-2-containing solution displayed increased levels of phosphorylated 44-45 kDa Cx43, assessed by western blotting. In addition, FGF-2 significantly upregulated phosphorylation of the PKC target serines 262 and 368 on Cx43 at intercalated disks, assessed using phosphospecific antibodies in immunolocalization and western blotting assays. Our data show that FGF-2, administered by perfusion, can alter the phosphorylation status of Cx43 at cardiomyocyte intercalated disks, and suggest a link between phosphorylation of Cx43 at specific PKC sites and FGF-2 cardioprotection.     

Connexin 43 phosphorylation and degradation are required for adipogenesis

Connexin-43 (Cx43) is a membrane phosphoprotein that mediates direct inter-cellular communication by forming gap junctions. In this way Cx43 can influence gene expression, differentiation and growth. Its role in adipogenesis, however, is poorly understood. In this study, we established that Cx43 becomes highly phosphorylated in early adipocyte differentiation and translocates to the plasma membrane from the endoplasmic reticulum. As preadipocytes differentiate, Cx43 phosphorylation declines, the protein is displaced from the plasma membrane, and total cellular levels are reduced via proteosomal degradation. Notably, we show that inhibiting Cx43 degradation or constitutively over-expressing Cx43 blocks adipocyte differentiation. These data reveal that transient activation of Cx43 via phosphorylation followed by its degradation is vital for preadipocyte differentiation and maturation of functional adipocytes.     

Protein kinase C-epsilon mediates phorbol ester-induced phosphorylation of connexin-43

We have used adenoviral vectors to express dominant negative variants of protein kinase C epsilon (PKCepsilon) or mitogen kinase kinase 1 (MKK1) to investigate their involvement in phorbol ester-induced connexin-43 (Cx43) phosphorylation in cardiomyocytes. Stimulation of cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) increased the fraction of the slower migrating (> or = 45 kDa) and more extensively phosphorylated Cx43 species. Expression of dominant negative MKK1 did not prevent the effect of PMA on Cx43 phosphorylation. Selective inhibition of PKCE significantly decreased baseline levels of Cx43 phosphorylation and the PMA-induced accumulation of > or = 45 kDa Cx43. Thus, production of the more extensively phosphorylated species of Cx43 in cardiomyocytes by PMA requires activation of PKCepsilon.