Hsin Lin Cheng

Bidirectional Regulation of p38 Kinase and c-Jun N-terminal Protein Kinase by Insulin-like Growth Factor-I

We have previously shown that insulin-like growth factor I (IGF-I) activation of the IGF-I receptor rescues SH-SY5Y human neuroblastoma cells from high glucose-mediated programmed cell death (PCD). In the current study, we further explored the potential points in the cell death cascade where IGF-I receptor activation may afford neuroprotection. As an initial step, we examined the effects of the PCD stimulus, high glucose, on stress-activated protein kinases, specifically the two mitogen-activated protein kinases p38 kinase and c-Jun N-terminal kinase (JNK). High glucose treatment activated the tyrosine phosphorylation of both p38 kinase and JNK in a dose- and time-dependent fashion. We next examined the effects of IGF-I on JNK and p38 kinase under normoglycemic and hyperglycemic conditions. IGF-I activated p38 kinase alone and had additive effects on glucose-induced p38 kinase phosphorylation. In contrast, IGF-I inhibited glucose activation of JNK phosphorylation and JNK activity. IGF-I also inhibited the glucose-induced nuclear translocation of JNK, but did not effect glucose-induced translocation of p38 kinase. Finally, IGF-I inhibition of JNK phosphorylation was blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, PD98059. Collectively, these data imply cross-talk between the mitogen-activated protein kinase pathway and JNK and suggest that IGF-I activation of mitogen-activated protein kinases interferes with JNK activation and protects cells from PCD.

Characterization of Insulin-Like Growth Factor-I and Its Receptor and Binding Proteins in Transected Nerves and Cultured Schwann Cells

Abstract: The insulin‐like growth factors (IGFs) are trophic factors whose growth‐promoting actions are mediated via the IGF‐I receptor and modulated by six IGF binding proteins (IGFBPs). In this study, we observed increased transcripts of both IGF‐I and IGF‐I receptor after rat sciatic nerve transection. Schwann cells (SCs) were the main source of IGF‐I and IGFBP‐5 immunoreactivity until 7 days after nerve transection, when invading macrophages in the distal nerve stumps were strongly IGF‐I positive. In vitro, IGF‐I promoted SC mitogenesis. Northern analysis revealed that SCs expressed IGF‐I receptor and IGFBP‐5. IGF‐I treatment increased the intensity of IGFBP‐5 without affecting gene expression. Des(1–3)IGF‐I, an IGF‐I analogue with low affinity for IGFBP, had no such effect. Incubation of recombinant human IGFBP‐5 with SC conditioned media revealed IGF‐I protection of IGFBP‐5 from proteolysis, implying the presence of an IGFBP‐5 protease in SC conditioned media. Collectively, these data support the concept that, in response to nerve injury, invading macrophages produce IGF‐I and SC express the IGF‐I receptor, to facilitate regeneration. This regenerative process may be augmented further by the ability of SC to secrete IGFBPs, which in turn may increase local IGF‐I bioavailability.

Insulin-like growth factor-I prevents caspase-mediated apoptosis in Schwann cells

Both neurons and glia succumb to programmed cell death (PCD) when deprived of growth factors at critical periods in development or following injury. Insulin-like growth factor-I (IGF-I) prevents apoptosis in neurons in vitro. To investigate whether IGF-I can protect Schwann cells (SC) from apoptosis, SC were harvested from postnatal day 3 rats and maintained in serum-containing media until confluency. When cells were switched to serum-free defined media (DM) for 12-72 h, they underwent PCD. Addition of insulin or IGF-I prevented apoptosis. Bisbenzamide staining revealed nuclear condensation and formation of apoptotic bodies in SC grown in DM alone, but SC grown in DM plus IGF-I had normal nuclear morphology. The phosphatidylinositol 3-kinase (PI 3-K) inhibitor LY294002 blocked IGF-I-mediated protection. Caspase-3 activity was rapidly activated upon serum withdrawal in SC, and the caspase inhibitor BAF blocked apoptosis. These results suggest that IGF-I rescues SC from apoptosis via PI 3-K signaling which is upstream from caspase activation.

IGF-I promotes Schwann cell motility and survival via activation of Akt.

We previously reported insulin-like growth factor-I (IGF-I) promotes Schwann cell (SC) motility and rescues SC from apoptosis induced by serum deprivation. This effect is mediated by phosphatidylinositol-3 (PI-3) kinase. In the current study, we examined the role of Akt, a downstream kinase of PI-3K, in SC motility and IGF-I mediated protection from apoptosis. IGF-I induces Akt phosphorylation at Ser473, an event which may be blocked by pretreatment with a PI-3K inhibitor, LY294002. In dominant negative K179M Akt (K179M) transfected SC, however, Akt is not activated in response to IGF-I. In addition, IGF-I is unable to promote SC motility and survival in K179M SC. These results suggest a critical role for Akt in IGF-I mediated motility and survival in SC.

Caveolin-1 Expression in Schwann Cells

Caveolae are non-clathrin-coated invaginations of the plasma membrane, which are present in most cell types. An integral component of caveolae is the caveolin family of related proteins, which not only forms the structural framework of caveolae, but also likely subserves its functional roles, including regulation of signal transduction and cellular transport, in particular, cholesterol trafficking. Although caveolae have been identified ultrastructurally in the peripheral nervous system (PNS), caveolin expression has not previously been studied. To date, three caveolin genes have been reported. Here, we show for the first time that caveolin-1 is expressed by Schwann cells (SC) as well as several SC-derived cell lines. Caveolin-1 is enriched in the buoyant, detergent-insoluble membranes of rat sciatic nerve (SN) and SC, a hallmark of the caveolar compartment. Caveolin-1 exists as both soluble and insoluble forms in rat SN and SC, and localizes to SC cytoplasm and abaxonal myelin. SC caveolin-1 decreases after axotomy, when SC revert to a premyelinating phenotype. We speculate that caveolin-1 may regulate signal transduction and/or cholesterol transport in myelinating SC.

IGF‐I Promotes Peripheral Nervous System Myelination

ABSTRACT: Insulin‐like growth factor‐I (IGF‐I) promotes the proliferation and differentiation of Schwann cells (SC). We use SC/dorsal root ganglion neuron (DRG) cocultures to examine the effects of IGF‐I on the interaction between axons and SC. As SC extend processes toward the axon in the presence of IGF‐I, these processes attach to and ensheath axons. Continued IGF‐I exposure leads to enhanced P0 expression and long‐term myelination. No myelination occurs in the absence of IGF‐I. These data imply that IGF‐I is critical not only for SC attachment and ensheathment of axons but also for long‐term myelination.

Insulin-like growth Factor-I (IGF-I) and IGF binding protein-5 in Schwann cell differentiation

Schwann cells (SCs) are the myelin producing cells of the peripheral nervous system. During development, SCs cease proliferation and differentiate into either a myelin‐forming or non‐myelin forming mature phenotype. We are interested in the role of insulin‐like growth factor‐I (IGF‐I) in SC development. We have shown previously SCs proliferate in response to IGF‐I in vitro. In the current study, we investigated the role of IGF‐I in SC differentiation. SC differentiation was determined by morphological criteria and expression of myelin proteins. Addition of 1 mM 8‐bromo cyclic AMP (cAMP) or growth on Matrigel matrix decreased proliferation and induced differentiation of SCs. IGF‐I enhanced both cAMP and Matrigel matrix‐induced SC differentiation, as assessed by both morphological criteria and myelin gene expression. Cultured SCs also express IGF binding protein‐5 (IGFBP‐5), which can modulate the actions of IGF‐I. We examined the expression of IGFBP‐5 during SC differentiation. Both cAMP and Matrigel matrix treatment enhanced IGFBP‐5 protein expression and cAMP increased IGFBP‐5 gene expression five fold. These findings suggest IGF‐I potentiates SC differentiation. The concomitant up‐regulation of IGFBP‐5 may play a role in targeting IGF‐I to SCs and thus increase local IGF‐I bioavailability. J. Cell. Physiol. 171:161–167, 1997.

Insulin-like growth factor-I and Bcl-XL inhibit c-jun N-terminal kinase activation and rescue Schwann cells from apoptosis

We previously reported that Schwann cells undergo apoptosis after serum withdrawal. Insulin‐like growth factor‐I, via phosphatidylinositol‐3 kinase, inhibits caspase activation and rescues Schwann cells from serum withdrawal‐induced apoptosis. In this study, we examined the role of c‐jun N‐terminal protein kinase (JNK) in Schwann cell apoptosis induced by serum withdrawal. Activation of both JNK1 and JNK2 was detected 1 h after serum withdrawal with the maximal level detected at 2 h. A dominant negative JNK mutant, JNK (APF), blocked JNK activation induced by serum withdrawal and Schwann cell apoptosis, suggesting JNK activation participates in Schwann cell apoptosis. Serum withdrawal‐induced JNK activity was caspase dependent and inhibited by a caspase 3 inhibitor, Ac‐DEVD‐CHO. Because insulin‐like growth factor‐I and Bcl‐XL are both Schwann cell survival factors, we tested their effects on JNK activation during apoptosis. Insulin‐like growth factor‐I treatment decreased both JNK1 and JNK2 activity induced by serum withdrawal. LY294002, a phosphatidylinositol‐3 kinase inhibitor, blocked insulin‐like growth factor‐I inhibition on JNK activation, suggesting that phosphatidylinositol‐3 kinase mediates the effects of insulin‐like growth factor‐I. Overexpression of Bcl‐XL also resulted in less Schwann cell death and inhibition of JNK activation after serum withdrawal. Collectively, these results suggest JNK activation is involved in Schwann cell apoptosis induced by serum withdrawal. Insulin‐like growth factor‐I and Bcl family proteins rescue Schwann cells, at least in part, by inhibition of JNK activity.

Immunohistochemical localization of insulin-like growth factor binding protein-5 in the developing rat nervous system.

The insulin-like growth factors (IGF-I and IGF-II) are peptides with both growth-promoting and insulin-like metabolic effects. The IGFs interact with and are modulated by a group of six IGF-binding proteins (IGFBP-1 through IGFBP-6). Previous studies have characterized IGFBP-5 and IGF-I gene expression in the developing nervous system. In the current study, cellular and tissue-specific distribution of IGFBP-5 protein was examined in the developing rodent nervous system using immunohistochemistry. Beginning with embryonic stage E12, IGFBP-5 immunoreactivity was observed in peripheral nerves. This pattern persisted through adulthood and was detected within Schwann cells and axons after postnatal day 16 (P16). IGFBP-5 immunoreactivity first appeared in the CNS at P16. Purkinje cells of the cerebellum were immunostained at P16, P32 and in the adult. IGFBP-5 immunoreactivity was also detected in several brain stem nuclei and their corresponding tracts as well as neuroglia. Nerve tracts and glia in the postnatal spinal cord were also immunopositive, however, spinal cord neurons were not stained. The current results, coupled with the known profile of IGF-I expression during nervous system development demonstrates the colocalization of IGF-I and IGFBP-5 in PNS, cerebellum, and brain stem.