Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function

During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.

A biologic function for an "orphan" messenger: D-myo-inositol 3,4,5,6-tetrakisphosphate selectively blocks epithelial calcium-activated chloride channels.

Inositol phosphates are a family of water-soluble intracellular signaling molecules derived from membrane inositol phospholipids. They undergo a variety of complex interconversion pathways, and their levels are dynamically regulated within the cytosol in response to a variety of agonists. Relatively little is known about the biological function of most members of this family, with the exception of inositol 1,4,5-trisphosphate. Specifically, the biological functions of inositol tetrakisphosphates are largely obscure. In this paper, we report that D-myo-inositol 3,4,5,6-tetrakisphosphate (D-Ins(3,4,5,6)P4) has a direct biphasic (activation/inhibition) effect on an epithelial Ca(2+)-activated chloride channel. The effect of D-Ins(3,4,5,6)P4 is not mimicked by other inositol tetrakisphosphate isomers, is dependent on the prevailing calcium concentration, and is influenced when channels are phosphorylated by calmodulin kinase II. The predominant effect of D-Ins(3,4,5,6)P4 on phosphorylated channels is inhibitory at levels of intracellular calcium observed in stimulated cells. Our findings indicate the biological function of a molecule hitherto considered as an "orphan" messenger. They suggest that the molecular target for D-Ins(3,4,5,6)P4 is a plasma membrane Ca(2+)-activated chloride channel. Regulation of this channel by D-Ins(3,4,5,6)P4 and Ca2+ may have therapeutic implications for the disease states of both diabetic nephropathy and cystic fibrosis.

Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1/Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca2+ influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca2+ entry is lacking in CD95-defective lpr lymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca2+ influx, activation of the transcriptional regulator NFAT, and IL-2 synthesis. The block of CRAC by sphingomyelinase metabolites adds a function to the repertoire of the CD95 receptor inhibiting T cell activation signals.

Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 μM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.

β-Amyloid peptide blocks the response of α7-containing nicotinic receptors on hippocampal neurons

Alzheimer's disease produces a devastating decline in mental function, with profound effects on learning and memory. Early consequences of the disease include the specific loss of cholinergic neurons in brain, diminished cholinergic signaling, and the accumulation of β-amyloid peptide in neuritic plaques. Of the nicotinic acetylcholine receptors at risk, the most critical may be those containing the α7 gene product (α7-nAChRs), because they are widespread, have a high relative permeability to calcium, and regulate numerous cellular events in the nervous system. With the use of whole-cell patch–clamp recording we show here that nanomolar concentrations of β-amyloid peptides specifically and reversibly block α7-nAChRs on rat hippocampal neurons in culture. The block is noncompetitive, voltage-independent, and use-independent and is mediated through the N-terminal extracellular domain of the receptor. It does not appear to require either calcium influx or G protein activation. β-Amyloid blockade is likely to be a common feature of α7-nAChRs because it applies to the receptors at both somato-dendritic and presynaptic locations on rat hippocampal neurons and extends to homologous receptors on chick ciliary ganglion neurons as well. Because α7-nAChRs in the central nervous system are thought to have numerous functions and recently have been implicated in learning and memory, impaired receptor function in this case may contribute to cognitive deficits associated with Alzheimer's disease.

The herpes simplex virus major regulatory protein ICP4 blocks apoptosis induced by the virus or by hyperthermia.

Cells infected with herpes simplex virus 1 (HSV-1) undergo productive or latent infection without exhibiting features characteristic of apoptosis. In this report, we show that HSV-1 induces apoptosis but has evolved a function that blocks apoptosis induced by infection as well as by other means. Specifically, (i) Vero cells infected with a HSV-1 mutant deleted in the regulatory gene alpha 4 (that encodes repressor and transactivating functions), but not those infected with wild-type HSV-1(F), exhibit cytoplasmic blebbing, chromatin condensation, and fragmented DNA detected as a ladder in agarose gels or by labeling free DNA ends with terminal transferase; (ii) Vero cells infected with wild-type HSV-1(F) or cells expressing the alpha 4 gene and infected with the alpha 4- virus did not exhibit apoptosis; (iii) fragmentation of cellular DNA was observed in Vero cells that were mock-infected or infected with the alpha 4- virus and maintained at 39.5 degrees C, but not in cells infected with wild-type virus and maintained at the same temperature. Wild-type strains of HSV-1 with limited extrahuman passages, such as HSV-1 (F), carry a temperature-sensitive lesion in the alpha 4 gene and at 39.5 degrees C only alpha genes are expressed. These results indicate that the product of the alpha 4 gene is able to suppress apoptosis induced by the virus as well by other means.

Conservation and function of the transcriptional regulatory protein Runt.

A phylogenetic approach was used to identify conserved regions of the transcriptional regulator Runt. Alignment of the deduced protein sequences from Drosophila melanogaster, Drosophila pseudoobscura, and Drosophila virilis revealed eight blocks of high sequence homology separated by regions with little or no homology. The largest conserved block contains the Runt domain, a DNA and protein binding domain conserved in a small family of mammalian transcription factors. The functional properties of the Runt domain from the D. melanogaster gene and the human AML1 (acute myeloid leukemia 1) gene were compared in vitro and in vivo. Electrophoretic mobility-shift assays with Runt/AML1 chimeras demonstrated that the different DNA binding properties of Runt and AML1 are due to differences within their respective Runt domains. Ectopic expression experiments indicated that proteins containing the AML1 Runt domain function in Drosophila embryos and that sequences outside of this domain are important in vivo.

An antiestrogen: a phosphotyrosyl peptide that blocks dimerization of the human estrogen receptor.

We have previously identified tyrosine-537 as a constitutively phosphorylated site on the human estrogen receptor (hER). A 12-amino acid phosphotyrosyl peptide containing a selected sequence surrounding tyrosine-537 was used to investigate the function of phosphotyrosine-537. The phosphotyrosyl peptide completely blocked the binding of the hER to an estrogen response element (ERE) in a gel mobility shift assay. Neither the nonphosphorylated tyrosyl peptide nor an unrelated phosphotyrosyl peptide previously shown to inhibit the signal transducers and activators of transcription factor (STAT) blocked binding of the hER to the ERE. The hER phosphotyrosyl peptide was shown by molecular sizing chromatography to dissociate the hER dimer into monomers. The hER specifically bound the 32P-labeled phosphotyrosyl peptide, indicating that the inhibition of ERE binding was caused by the phosphotyrosyl peptide binding directly to the hER and blocking dimerization. These data suggest that the phosphorylation of tyrosine-537 is a necessary step for the formation of the hER dimer. In addition, we propose that the dimerization of the hER occurs by a previously unrecognized Src homology 2 domain (SH2)-like phosphotyrosyl coupling mechanism. Consequently, the phosphotyrosyl peptide represents a class of antagonists that inhibits estrogen action by a mechanism other than interacting with the receptor's hormone binding site.

Overexpression of the c-Myc oncoprotein blocks the growth-inhibitory response but is required for the mitogenic effects of transforming growth factor beta 1.

One of the more intriguing aspects of transforming growth factor beta 1 (TGF beta 1) is its ability to function as both a mitogenic factor for certain mesenchymal cells and a potent growth inhibitor of lymphoid, endothelial, and epithelial cells. Data are presented indicating that c-myc may play a pivotal role in both the mitogenic and antiproliferative actions of TGF beta 1. In agreement with previous studies using C3H/10T1/2 fibroblasts constitutively expressing an exogenous c-myc cDNA, we show that AKR-2B fibroblasts expressing a chimeric estrogen-inducible form of c-myc (mycER) are able to form colonies in soft agar in the presence of TGF beta 1 only when c-myc is activated by hormone. Whereas these findings support a synergistic role for c-myc in mitogenic responses to TGF beta 1, we also find that c-myc can antagonize the growth-inhibitory response to TGF beta 1. Mouse keratinocytes (BALB/MK), which are normally growth-arrested by TGF beta 1, are rendered insensitive to the growth-inhibitory effects of TGF beta 1 upon mycER activation. This ability of mycER activation to block TGF beta 1-induced growth arrest was found to occur only when the fusion protein was induced with hormone in the early part of G1. Addition of estradiol late in G1 had no suppressive effect on TGF beta 1-induced growth inhibition.