Bethel Stannard

Muscle-specific inactivation of the IGF-I receptor induces compensatory hyperplasia in skeletal muscle.

During the development of skeletal muscle, myoblasts withdraw from the cell cycle and differentiate into myotubes. The insulin-like growth factors IGF-I and IGF-II, through their cognate tyrosine kinase receptor (IGF-I receptor), are known to play a role in this process. After withdrawal of myoblasts from the cell cycle, IGF-I promotes muscle differentiation by inducing the expression or activity of myogenic regulatory factors (MyoD, myogenin) and effectors (p21). However, little is known about the intracellular mechanisms by which the IGF-I system regulates these factors during the process of myogenesis. Here we show that MKR mice, which express a dominant negative IGF-I receptor specifically in skeletal muscle, have marked muscle hypoplasia from birth to 3 weeks of age. This hypoplasia occurs concomitantly with a decrease in ERK immunoreactivity levels and decreases in MyoD and myogenin expression. BrdU immunocytochemistry showed a compensatory hyperplasia as MKR mice grew to adulthood. Interestingly, hyperplasia occurred concomitantly with an increase in p38, MyoD, myogenin, and p21 immunoreactivity levels, as well as a decrease in Twist levels. These findings suggest that regulation of these cellular elements by IGF-I may play a role in the development and differentiation of skeletal muscle in vivo.

Experimental Diabetes Increases Insulinlike Growth Factor I and II Receptor Concentration and Gene Expression in Kidney

Insulinlike growth factor I (IGF-I) is a mitogenic hormone with important regulatory roles in growth and development. One of the target organs for IGF-I action is the kidney, which synthesizes abundant IGF-I receptors and IGF-I itself. To study the involvement of IGF-I and the IGF-I receptor in the development of nephropathy, one of the major complications of diabetes mellitus, we measured the expression of these genes in the kidney and in other tissues of the streptozocin-induced diabetic rat. The binding of 125I-labeled IGF-I to crude membranes was measured in the same tissues. We observed a 2.5-fold increase in the steady-state level of IGF-I–receptor mRNA in the diabetic kidney, which was accompanied by a 2.3-fold increase in IGF-I binding. In addition to this increase in IGF-I binding to the IGF-I receptor, there was also binding to a lower-molecular-weight material that may represent an IGF-binding protein. No change was detected in the level of IGF-I–peptide mRNA. Similarly, IGF-II–receptor mRNA levels and IGF-II binding were significantly increased in the diabetic kidney. IGF-I– and IGF-II-receptor mRNA levels and IGF-I and IGF-II binding returned to control values after insulin treatment. Because the IGF-I receptor is able to transduce mitogenic signals on activation of its tyrosine kinase domain, we hypothesize that, among other factors, high levels of receptor in the diabetic kidney may also be involved in the development of diabetic nephropathy. Increased IGF-II–receptor expression in the diabetic kidney may be important for the intracellular transport and packaging of lysosomal enzymes, although a role for this receptor in signal transduction cannot be excluded. Finally, the possible role of IGF-binding proteins requires further study.

Mice transgenically overexpressing sulfonylurea receptor 1 in forebrain resist seizure induction and excitotoxic neuron death

The ability of the sulfonylurea receptor (SUR) 1 to suppress seizures and excitotoxic neuron damage was assessed in mice transgenically overexpressing this receptor. Fertilized eggs from FVB mice were injected with a construct containing SUR cDNA and a calcium-calmodulin kinase IIα promoter. The resulting mice showed normal gross anatomy, brain morphology and histology, and locomotor and cognitive behavior. However, they overexpressed the SUR1 transgene, yielding a 9- to 12-fold increase in the density of [3H]glibenclamide binding to the cortex, hippocampus, and striatum. These mice resisted kainic acid-induced seizures, showing a 36% decrease in average maximum seizure intensity and a 75% survival rate at a dose that killed 53% of the wild-type mice. Kainic acid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons or expression of heat shock protein 70, whereas wild-type mice lost 68–79% of pyramidal neurons in the CA1–3 subfields and expressed high levels of heat shock protein 70 after kainate administration. These results indicate that the transgenic overexpression of SUR1 alone in forebrain structures significantly protects mice from seizures and neuronal damage without interfering with locomotor or cognitive function.

INSULIN-LIKE GROWTH FACTOR-I INHIBITS THE STRESS-ACTIVATED PROTEIN KINASE/C-JUN N-TERMINAL KINASE

The pathways involved in the cellular responses to the insulin-like growth factors (IGFs) are numerous and vary according to cell type. Following activation of the IGF-I receptor, the mitogen-activated protein kinase and phosphatidylinositide 3′-kinase (PI3′K) pathways are activated and result in cellular proliferation and inhibition of apoptosis. In this study, we analyzed the IGF-I effect on the stress-activated protein kinase/c-Jun N-terminal kinase (JNK) activity using human embryonic kidney 293 cells, 293 cells transiently expressing hemagglutinin-JNK, and 293 cells stably expressing a hemagglutinin-JNK transgene. In all cell types, endogenous or transfected JNK activity was strongly stimulated by anisomycin or tumor necrosis factor-α, and 10 nm IGF-I pretreatment suppressed the induced JNK activity. To determine whether the effect of IGF-I on JNK activity involves the mitogen-activated protein kinase or PI3′K pathway, we used the specific MEK1 inhibitor PD098059 and the PI3′K inhibitor LY 294002. PD098059 did not alter the IGF-I suppressive effect on stressor-induced JNK activity, but LY 294002 suppressed the IGF-I effect. Moreover, in transiently transfected parental 293 cells expressing dominant-negative Akt, anisomycin-increased JNK activity was not suppressed by pretreatment with IGF-I. Our results demonstrate that the action of IGF-I on JNK in these cells is via PI3′K and Akt.

In vitro and in vivo responses to short-term recombinant human insulin-like growth factor-1 (IGF-I) in a severely growth-retarded girl with ring chromosome 15 and deletion of a single allele for the type 1 IGF receptor gene

Patients with single allele defects in the gene encoding the type 1 IGF receptor have been reported to have growth failure, but fibroblasts from affected patients have not exhibited insensitivity to the effects of IGF‐I in vitro. The in vitro and in vivo responses to short‐term recombinant human IGF‐I (rhIGF‐I) in a severely growth‐retarded girl with ring chromosome 15 and deletion of a single allele for the type 1 IGF receptor gene have been investigated.

Protection against hypoxic-ischemic injury in transgenic mice overexpressing Kir6.2 channel pore in forebrain

The role of the K-ATP channel pore-forming subunit Kir6.2 on protection from cerebral hypoxic-ischemic injury was assessed in transgenic mice overexpressing normal Kir6.2 or a dominant negative form (AFA) of this subunit in the forebrain. The resulting mice overexpress either the Kir6.2 or the AFA transgene mainly in the cerebral cortex and hippocampus. The Kir6.2 transgenic mice are resistant to hypoxic-ischemic injury showing a decreased region of cortical damage as compared to the dominant negative AFA and the wild-type mice. Moreover, the overexpression of Kir6.2 allowed an important silencing of the neurons present in forebrain regions thus protecting them from ischemic injury. Interestingly, the phenotype observed in Kir6.2 transgenic mice was observed without increased sulfonylurea binding. Taken together, these results indicate that the transgenic overexpression of Kir6.2 in forebrain significantly protects mice from hypoxic-ischemic injury and neuronal damage seen in stroke.

Replacement of tyrosine 1251 in the carboxyl terminus of the insulin-like growth factor-I receptor disrupts the actin cytoskeleton and inhibits proliferation and anchorage-independent growth.

Insulin-like growth factor (IGF)-I signaling through the IGF-I receptor modulates cellular adhesion and proliferation and the transforming ability of cells overexpressing the IGF-I receptor. Tyrosine phosphorylation of intracellular proteins is essential for this transduction of the IGF-I-induced mitogenic and tumorigenic signals. IGF-I induces specific cytoskeletal structure and the phosphorylation of proteins in the associated focal adhesion complexes. The determination of the exact pathways emanating from the IGF-I receptor that are involved in mediating these signals will contribute greatly to the understanding of IGF-I action. We have previously shown that replacement of tyrosine residues 1250 and 1251 in the carboxyl terminus of the IGF-I receptor abrogates IGF-I-induced cellular proliferation and tumor formation in nude mice. In this study, replacement of either tyrosine 1250 or 1251 similarly reduces the cells ability to grow in an anchorage-independent manner. The actin cytoskeleton and cellular localization of vinculin are disrupted by replacement of tyrosine 1251. Tyrosine residues 1250 and 1251 are not essential for tyrosine phosphorylation of two known substrates; insulin receptor substrate-1 and SHC, nor association of known downstream adaptor proteins to these substrates. In addition, these mutant IGF-I receptors do not affect IGF-I-stimulated p42/p44 mitogen-activated protein kinase activation or phosphatidylinositol (PI) 3′-kinase activity. Thus, it appears that in fibroblasts expressing tyrosine 1250 and 1251 mutant IGF-I receptors, the signal transduction pathways impacting on mitogenesis and tumorigenesis do not occur exclusively through the PI 3′-kinase or mitogen-activated protein kinase pathways.

Glycosylation and posttranslational processing of thyroid-stimulating hormone: clinical implications.

Publisher Summary This chapter discusses clinical implications of glycosylation and posttranslational processing of thyroid-stimulating hormone (TSH). TSH is a glycoprotein hormone of molecular weight 28,000 which is composed of two noncovalently linked subunits, α and β . It is chemically related to the pituitary gonadotropins, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and to chorionic gonadotropin (CG). Elevated serum α /TSH ratios have been described in patients with TSH-secreting pituitary tumors. The excess production from such tumors has been of value in differentiating patients with neoplastic from non-neoplastic causes of TSH-induced hyperthyroidism. In addition, isolated production of α subunit, without concomitant production of TSH or gonadotropins, by certain pituitary adenomas and other malignant tumors is demonstrated. Elevated serum TSH- β /TSH ratios are described in two patients, one with an enlarged thyroid and one with an enlarged pituitary. The TSH- β in both cases have large molecular weight, display immunologic properties different from standard TSH- β , and is unresponsive to thyrotropin releasing hormone. Although this unusual form of TSH- β has been partially characterized, its significance remains unknown. The chapter also describes unusual forms of human TSH with decreased bioactivity. One apparently normal subject was found to have a high-molecular-weight form of TSH with normal receptor-binding properties but decreased bioactivity. Such forms can result from aggregated or protein-bound TSH caused by abnormalities of glycosylation, similar to those noted after tunicamycin treatment.

Precursor—product relationships in the biosynthesis and secretion of thyrotropin and its subunits by mouse thyrotropic tumor cells

TSH is composed of two non-covalently linked, glycosylated subunits, an (Y subunit that is virtually identical to that in LH, FSH, and CG as well as a unique p subunit that confers biologic specificity [ 1 ] . Although heterogeneous molecular weight forms of TSH as well as other glycoprotein hormones and their subunits have been recognized [2-61, the precursorproduct relationships among various forms have not yet been elucidated. In this report we describe such relationships in the de novo biosynthesis of TSH using primary cultures of dispersed mouse pituitary thyrotropic tumor cells.

Regulation of Insulin-Like Growth Factor I Receptor Gene Expression in Normal and Pathological States

Most of the biological actions of insulin-like growth factor I (IGF-I)/somatomedin C are initiated by its binding to the IGF-I receptor, a heterotetrameric glycoprotein structurally related to the insulin receptor1. The presence of IGF-I receptors in most body tissues suggests that it mediates many different effects. Indeed, IGF-I has been shown to be involved not only in endocrine functions, such as the mediation of growth hormone’s effect on longitudinal growth, but it is also involved in many autocrine/paracrine systems at the local tissue level2,3. These biological actions include both short-term, metabolic effects (similar in nature to those stimulated by insulin) as well as long term, growth promoting actions. It is not surprising then, that the IGF- I receptor should be able to respond to various tissue — and development- specific stimuli. To study the expression of the IGF-I receptor gene in both physiological and pathological conditions in a convenient animal model, we undertook the cloning of rat IGF-I receptor cDNAs.