Pyk2 and FAK regulate neurite outgrowth induced by growth factors and integrins

Integration of signalling pathways initiated by receptor tyrosine kinases and integrins is essential for growth-factor-mediated biological responses. Here we show that co-stimulation of growth-factor receptors and integrins activates the focal-adhesion kinase (FAK) family to promote outgrowth of neurites in PC12 and SH-SY5Y cells. Pyk2 and FAK associate with adhesion-based complexes that contain epidermal growth factor (EGF) receptors, through their carboxy- and amino-terminal domains. Expression of the C-terminal domain of Pyk2 or of FAK is sufficient to block neurite outgrowth, but not activation of extracellular-signal-regulated kinase (ERK). Moreover, activation and autophosphorylation of Pyk2/FAK, as well as of effectors of their adhesion-targeting domains, such as paxillin, are important for propagation of signals that control neurite formation. Thus, Pyk2/FAK have important functions in signal integration proximal to integrin/growth-factor receptor complexes in neurons.     

Nutrition, insulin, insulin-like growth factors and cancer.

The incidence of colon, pancreatic, and kidney cancers, as well as aggressive prostate cancer in men, and breast and endometrial cancer in women is invariably high in Western countries. Nutritional and related factors have been typically implicated. This review presents a model integrating nutrition, insulin and IGF-1 physiology ("bioactive" IGF-1), and carcinogenesis based on the following: (1) insulin and the IGF-1 axis function in an integrated fashion to promote cell growth and survival; (2) chronic exposure to these growth properties enhances carcinogenesis; (3) factors that influence bioactive IGF-1 will affect cancer risk. The model presented here summarizes the data that chronic exposure to high levels of insulin and IGF-1 may mediate many of the risk factors for some cancers that are high in Western populations. This hypothesis may help explain some of the epidemiologic patterns observed for these cancers, both from a cross-national perspective and within populations. Of particular importance is that some of relevant factors are modifiable through nutritional and lifestyle interventions. Out of a variety of perspectives presented, nutritional manipulation through the insulin pathway may be more feasible than attempting to influence total IGF-1 concentrations, which are determined largely by growth hormone. Further study is required to test these conclusions.     

Stimulation by melanocortins of neurite outgrowth from spinal and sensory neurons in vitro

Using ELISAs for B-50/GAP43 and neurofilament (NF), we tested ACTH(1–24), -MSH, ACTH(4–10), and an ACTH(4–9) analogue (ORG2766) for their ability to induce sprouting and neuritogenesis from spinal and sensory neurons. Dissociated fetal rat spinal cord neurons or neonatal rat dorsal root ganglion (DRG) cells were cultured with peptide and assayed after 24, 48, or 96 h. In spinal neurons, -MSH and ACTH(1–24) induced the expression of B-50 dose dependently. After 24 h -MSH had a stimulatory effect (from 10 nM onwards), with a maximum at 100 μM (36% increase). After 96 h the maximal effect of 100 μM -MSH on B-50/GAP43 was lower (19%). ACTH(1–24) (100 μM) stimulated B-50/GAP43 by 19%. Neurofilament levels (96 h) were elevated maximally by 64% at 100 μM -MSH. In DRG neurons a bell-shaped dose-response curve was found for -MSH, the maximal effect being observed after 48 h at 100 nM: 54% for B-50/GAP43 and 22% for NF. In both culture systems neither ACTH(4–10) nor ORG2766 was effective. We conclude that -MSH stimulates the expression of B-50/GAP43 (sprouting) and the formation of NF (neurite elongation) and may therefore be considered a neurotrophic factor.     

Organization of the human genes for insulin-like growth factors I and II

Recently, we have reported the isolation of cDNAs encoding the precursors of insulin-like growth factors I and II (IGF-I and II) [(1983) Nature 306, 609-611; (1985) FEBS Lett. 179, 243-246. These cDNAs were employed as specific probes to detect and isolate the corresponding genes from human cosmid DNA libraries. Three cosmids were detected, together containing the entire cDNA sequence of IGF-I, and one cosmid containing the sequence of IGF-II cDNA. Southern blot hybridization, physical mapping and nucleotide sequence analysis of these cosmids revealed that the IGF-I and -II genes have a discontinous structure. The IGF-I gene contains at least four exons spanning a region of probably more that 45 kilobasepairs (kb), while the IGF-II gene consists of at least five exons, spanning a region of 16 kb.     

Control of growth cone motility and neurite outgrowth by SPIN90

SPIN90 is an F-actin binding protein thought to play important roles in regulating cytoskeletal dynamics. It is known that SPIN90 is expressed during the early stages of neuronal development, but details of its localization and function in growth cones have not been fully investigated. Our immunocytochemical data show that SPIN90 is enriched throughout growth cones and neuronal shafts in young hippocampal neurons. We also found that its localization correlates with and depends upon the presence of F-actin. Detailed observation of primary cultures of hippocampal neurons revealed that SPIN90 knockout reduces both growth cone areas and in the numbers of filopodia, as compared to wild-type neurons. In addition, total neurite length, the combined lengths of the longest (axonal) and shorter (dendritic) neurites, was smaller in SPIN90 knockout neurons than wild-type neurons. Finally, Cdc42 activity was down-regulated in SPIN90 knockout neurons. Taken together, our findings suggest that SPIN90 plays critical roles in controlling growth cone dynamics and neurite outgrowth.     

Simultaneous isolation of insulin-like growth factors I and II from adult sheep serum

Ovine insulin-like growth factors I and II (oIGF-I and oIGF-II) have been purified from adult sheep serum. oIGF-II-like receptor-binding activity and IGF-I-like immunoactivity were enriched on SP-Sephadex C-25, then purified using HPLC in the presence of a variety of counter ions. IGF-I- and IGF-II-like activities were separated using HPLC in the presence of 0.2% tetrabutylammonium phosphate at pH 7.0. The final recovery of oIGF-I was 82.6 micrograms from 3.2 litres of adult sheep serum (a yield of 17.6%), and the recovery of oIGF-II was 388 micrograms (a yield of 13.3%). Both IGF preparations were considered to be homogeneous as judged by single sharp peaks during analytical HPLC, and unique N-terminal amino acid sequences. Purified ovine IGFs had molecular weights similar to that of other IGFs (approximately 7000), and the first 30 N-terminal amino acids of both peptides were identical to their human counterparts. The isoelectric points of oIGF-I (pI approximately 8.2) and oIGF-II (pI approximately 6.8) were similar to those of human (h) IGFs (hIGF-I pI approximately 8.2; hIGF-II pI approximately 6.5), and the overall amino acid content of the ovine IGFs was also similar to that of IGFs from other species. oIGF-II preparations from fetal sheep and from adult sheep appeared to be identical. The isolation procedure represents one of general utility that can be easily modified to facilitate the isolation of recombinant IGFs from culture fluid.     

Extracellular matrix and neurite outgrowth.

The complex relationship between neuronal cells and the extracellular matrix molecules with which they interact both positively and negatively is currently being investigated on many fronts. Major areas of experimental emphasis include the characterization of an increasing number of extracellular matrix and cell surface associated molecules, the identification of receptors for these molecules, and the analysis of the function of extracellular matrix molecules with respect to neuronal process outgrowth.     

Immunological relationships between receptors for insulin and insulin-like growth factor I. Evidence for structural heterogeneity of insulin-like growth factor I receptors involving hybrids with insulin receptors.

The receptors for insulin and insulin-like growth factor-I (IGF-I) are closely related in primary sequence and overall structure. We have examined the immunological relationships between these receptors by testing the reactivity of anti-(insulin receptor) monoclonal antibodies with IGF-I receptors in various tissues and cell lines. Antibodies for six distinct epitopes reacted with a subfraction of IGF-I receptors, as shown by inhibition of 125I-IGF-I binding, precipitation of 125I-IGF-I-receptor complexes or immunodepletion of receptor from tissue extracts before binding assays. Both immunoreactive and non-immunoreactive subfractions displayed the expected properties of 'classical' IGF-I receptors, in terms of relative affinities for IGF-I and insulin. The proportion of total IGF-I receptors which was immunoreactive varied in different cell types, being approx. 40% in Hep G2 cells, 35-40% in placental membranes and 75-85% in IM-9 cells. The immunoreactive fraction was somewhat higher in solubilized receptors than in the corresponding intact cells or membranes. A previously described monoclonal antibody, alpha-IR-3, specific for IGF-I receptors, inhibited IGF-I binding by more than 80% in all preparations. When solubilized placental receptors were pretreated with dithiothreitol (DTT) under conditions reported to reduce intramolecular (class I) disulphide bonds, the immunoreactivity of IGF-I receptors was abolished although total IGF-I binding was little affected. Under the same conditions insulin receptors remained fully immunoreactive. When solubilized receptor preparations were fractionated by gel filtration, both IGF-I and insulin receptors ran as symmetrical peaks of identical mobility. After DTT treatment, the IGF-I receptor was partially converted to a lower molecular mass form which was not immunoreactive. The insulin receptor peak showed a much less pronounced skewing and remained fully immunoreactive in all fractions. It is concluded that the anti- (insulin receptor) antibodies do not react directly with IGF-I receptor polypeptide, and that the apparent immunoreactivity of a subfraction of IGF-I receptors reflects their physical association with insulin receptors, both in cell extracts and in intact cells. The most likely basis for this association appears to be a 'hybrid' receptor containing one half (alpha beta) of insulin receptor polypeptide and the other (alpha' beta') of IGF-I receptor polypeptide within the native (alpha beta beta' alpha') heterotetrameric structure.     

Control of neurite outgrowth and growth cone motility by phosphatidylinositol-3-kinase

Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.     

Further characterization of human basic-somatomedin: comparison with insulin-like growth factors I and II

Our basic-somatomedin (SM) was further compared with insulin-like growth factors (IGF) I and II. Basic-SM and IGF revealed similar sulfation factor (SF) activity in cartilage, insulin-like activity(ILA) in adipocytes, and receptor binding activity to adipocytes and placental cell membranes.IGF-II revealed less SF activity but more ILA than basic-SM. Comparison of SM and insulin in terms of ILA and binding activity to adipocytes suggested that adipocytes have separate insulin and SM receptors and that the ILA of SM is mediated through the SM receptors. These studies also suggest that the receptors for acidic-neutral group of SM mediate the ILA of SM, whereas the growth promoting effects of SM are mediated via receptors for the basic group of SM.     

Stimulation by insulin-like growth factors is required for cellular transformation by type beta transforming growth factor

Medium conditioned by BRL-3A cells, a known source of insulin-like growth factor II (IGF-II), induced phenotypic transformation (anchorage-independent proliferation) of mouse BALB/c 3T3 fibroblasts but not rat NRK-49F fibroblasts, in the presence of 10% calf serum. A specific radioreceptor assay and a bioassay indicated that BRL-3A conditioned medium contained 0.5-1 ng/ml of type beta transforming growth factor (beta TGF). Purified IGF-II and beta TGF acting together reconstituted the transforming activity of BRL-3A conditioned medium on BALB/c 3T3 cells. Insulin was 5-10% as potent as IGF-II in supporting the transforming action of beta TGF on BALB/c 3T3 cells. NRK-49F cells were phenotypically transformed by beta TGF in the presence of EGF and 10% calf serum as the sole source of IGFs. However, transformation of NRK-49F cells under these conditions was inhibited by addition of purified IGF-binding protein. Addition of an excess of IGF-II prevented the inhibitory action of IGF-binding protein. The different sensitivity of the two cell lines to IGFs was correlated with lower levels of type I IGF receptor and higher levels of type II IGF receptor in NRK-49F cells as compared with BALB/c 3T3 cells. The results suggest that cellular stimulation by IGFs is a prerequisite for transformation of rodent fibroblasts by beta TGF. We propose that transformation of fibroblasts by beta TGF requires concomitant stimulation by the set of growth factors that support normal cell proliferation.     

Distinct biologically active receptors for insulin, insulin-like growth factor I, and insulin-like growth factor II in cultured skeletal muscle cells

The expression of insulin-like growth factor (IGF) receptors at the cell surface and the changes in IGF responsiveness during differentiation were studied in the L6 skeletal muscle cell line. Throughout the entire developmental sequence, distinct receptors for IGF I and IGF II that differed in structure and peptide specificity could be demonstrated. During differentiation, both 125I-IGF I and 125I-IGF II binding to the L6 cells decreased as a result of a 3-4-fold reduction in receptor number, whereas 125I-insulin binding increased. Under nonreducing conditions, disuccinimidyl suberate cross-linked 125I-IGF I and 125I-IGF II to two receptor complexes with apparent Mr greater than 300,000 (type I) and 220,000 (type II). Under reducing conditions, the apparent molecular weight of the type I receptor changed to Mr 130,000 (distinct from the 120,000 insulin receptor) and the type II receptor changed to 250,000. IGF I and IGF II both stimulated 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake in the L6 cells with a potency close to that of insulin, apparently through interaction with their own receptors. The stimulatory effects of IGF II correlated with its affinity for the type II but not the type I IGF receptor, as measured by inhibition of affinity labeling, whereas the effects of IGF I correlated with its ability to inhibit labeling of the type I receptor. In spite of the decrease in type I and type II receptor number, stimulation of 2-deoxy-glucose and alpha-aminoisobutyric acid uptake by the two IGFs increased during differentiation.     

Differential regulation of signaling pathways for insulin and insulin-like growth factor I.

The insulin receptor (IR) and the insulin-like growth factor receptor I (IGF-IR) have different functions in cell growth, apoptosis, differentation, and transformation. Although some of these differences may be explained by the relative level of receptor expression and receptor structure (alpha and beta subunits), they may also be attributed to differences in intracellular signals generated by insulin and IGF-I. The presence of hybrid receptors (IR alphabeta subunits and IGF-IR alphabeta subunits) making up the heterotetramers has added a new dimension to our understanding of the functional roles of these receptors. However, to date the results of efforts to understand the differences between these two closely related receptors have indicated mostly similarities. For example, both receptors utilize IRS-1/IRS-2 and Shc as immediate downstream adaptors, leading to activation of the Ras, Raf, ERK kinases and PI-3 kinase pathways. We have used the yeast two hybrid system to identify proteins which bind to the activated IGF-IR but not to the IR. The cytoplasmic domain of the IGF-IR was used to screen a human fetal brain library and two isoforms of the 14-3-3 family were identified. 14-3-3 proteins are a highly conserved family of proteins which have recently been shown to interact with other components of the mitogenic and apoptotic signaling pathways, including Raf, BAD, Bcr/Bcr-Abl, middle-T antigen, Ksr, PKC, PI-3 kinase, ASK1 kinase, and cdc25C phosphatase. We also identified human Grb10, an adaptor protein with SH2 domain associated with the IGF-IR beta subunit. Smith's laboratory showed that Grb10 preferentially binds to the IR in intact cells. Using the interaction trap screen (active cytoplasmic domain of the IGF-IR) 55PIK and SOCS-2 proteins were also identified. However, 55PIK and SOCS-2 also interact with the IR in the yeast two hybrid system. These studies raise the possibility that 14-3-3 and Grb10 may play a role in insulin and IGF-I signal transduction and may underlie the observed differences.     

In vivo regulation of cyclic AMP phosphodiesterase in Xenopus oocytes. Stimulation by insulin and insulin-like growth factor 1

Cyclic AMP phosphodiesterase activity was measured in vivo after microinjection of [3H]cAMP into intact Xenopus oocytes. This activity was inhibited by extracellular application of methylxanthines, and the dose-dependent inhibition of phosphodiesterase activity correlated with the abilities of isobutylmethylxanthine and theophylline to inhibit oocyte maturation induced by progesterone, with IC50 values of approximately 0.3 and 1.5 mM, respectively. Insulin stimulated in vivo phosphodiesterase activity measured after microinjection of 200 microM [3H]cAMP in a time- and dose-dependent fashion without affecting phosphodiesterase activity measured after microinjection of 2 microM [3H]cAMP. Although progesterone alone had no effect on in vivo phosphodiesterase activity, low concentrations of progesterone (0.01 microM) accelerated the time course of insulin stimulation of both phosphodiesterase activity and oocyte maturation. The EC50 for stimulation of in vivo phosphodiesterase activity by insulin correlated with the IC50 for inhibition of oocyte membrane adenylate cyclase activity measured in vitro (2 and 4 nM, respectively). Twenty-fold higher concentrations of insulin were required to stimulate oocyte maturation. In contrast, insulin-like growth factor 1 stimulated in vivo phosphodiesterase, inhibited in vitro adenylate cyclase, and induced oocyte maturation at concentrations of 0.3-1.0 nM. These results demonstrate a dual regulation of oocyte phosphodiesterase and adenylate cyclase by insulin and insulin-like growth factor 1.     

Receptors for insulin-like growth factors I and II in developing embryonic mouse limb bud

Insulin-like growth factors (IGF) or somatomedins (SM) have been classically defined as promoting the actions of growth hormone in skeletal growth. IGF is divided into two groups, IGF-I and II, and are presumed to act via IGF type I (higher affinity for IGF-I and II and very low affinity for insulin) and II (higher affinity for IGF-II than I and no affinity for insulin) receptors, respectively. Recently, a switchover role of IGF-II to I during fetal to adult growth has been suggested. We have investigated the possible transitional role of IGF-II to I in a developing mouse embryonic limb bud organ culture model. In this in vitro system, limb bud develops from the blastoma stage to a well-differentiated cartilage tissue. Both IGF type I and II receptors were found to be present in limb buds at all stages of differentiation. Type I receptor decreased with differentiation while Type II receptor increased. The effect of IGF-I on [3H]thymidine and [35S]sulfate uptake by the tissue increased with differentiation while the effect of IGF-II on [3H]thymidine uptake of the undifferentiated tissue was abolished with differentiation of the tissue. The increase of the IGF-I response with decreased type I receptor may reflect an altered receptor sensitivity (occupancy) during differentiation. The decrease of the IGF-II response with increased type II receptor with differentiation may on the other hand suggest that IGF-II in differentiated tissue no longer acts as a classical growth factor. These results tend to support the hypothesis of the switchover role of IGF-I and II during fetal and adult growth, however, confirmation of the precise role of IGF-I and II in biological growth may have to wait until further studies clarifying the significance of the increased IGF type II receptor in differentiated tissue are made.     

Stimulation of glucose transport in osteoblastic cells by parathyroid hormone and insulin-like growth factor I

Insulin and parathyroid hormone (PTH) regulate glucose metabolism in bone cells. In order to differentiate between the effects of these hormones and to compare the potency of insulin with that of insulin-like growth factor (IGF) I, we treated rat bone-derived osteoblastic (PyMS) cells for different time periods and at different concentrations with insulin, IGF I, or PTH, and measured [1-(14)C]-2-deoxy-D-glucose (2DG) uptake and incorporation of D-[U-(14)C] glucose into glycogen. 2DG uptake was Na-independent with an apparent affinity constant (K (M)) of ~2 mmol/l. Expression of the high affinity glucose transporters (GLUT), GLUT1 and GLUT3 but not of GLUT4, was found by Northern and Western analysis. Similar to the findings with primary rat osteoblasts, but distinct from those in rat fibroblasts, 2DG uptake and glycogen synthesis were increased in this cell line after exposure to low concentrations (0.1 nmol/l and above) of PTH. IGF I at low doses (0.3 nmol/l and above) or insulin at higher doses (1 nmol/l and above) stimulated 2DG uptake and [(3)H] thymidine incorporation into DNA. 2DG transport was enhanced already after 30 min of IGF I treatment whereas the effect of PTH became significant after 6 h. It is concluded that IGF I rather than insulin may be a physiological regulator of 2DG transport and glycogen synthesis in osteoblasts.