Neurite Growth and Polarization on Vitronectin Substrate after in Vitro Trauma is not Enhanced after IGF Treatment.

Following traumatic brain injuries (TBI), insulin-like growth factor (IGF) is cortically widely upregulated. This upregulation has a potential role in the recovery of neuronal tissue, plasticity, and neurotrophic activity, though the molecular mechanisms involved in IGF regulation and the exact role of IGF after TBI remain unclear. Vitronectin (VN), an extracellular matrix (ECM) molecule, has recently been shown to be of importance for IGF-mediated cellular growth and migration. Since VN is downregulated after TBI, we hypothesized that insufficient VN levels after TBI impairs the potential beneficial activity of IGF. To test if vitronectin and IGF-1/IGFBP-2 could contribute to neurite growth, we cultured hippocampal neurons on ± vitronectin-coated coverslips and them treated with ± IGF-1/IGF binding protein 2 (IGFBP-2). Under same conditions, cell cultures were also subjected to in vitro trauma to investigate differences in the posttraumatic regenerative capacity with ± vitronectin-coated coverslips and with ± IGF-1/IGFBP-2 treatment. In both the control and trauma situations, hippocampal neurons showed a stronger growth pattern on vitronectin than on the control substrate. Surprisingly, the addition of IGF-1/IGFBP-2 showed a decrease in neurite growth. Since neurite growth was measured as the number of neurites per area, we hypothesized that IGF-1/IGFBP-2 contributes to the polarization of neurons and thus induced a less dense neurite network after IGF-1/IGFBP-2 treatment. This hypothesis could not be confirmed and we therefore conclude that vitronectin has a positive effect on neurite growth in vitro both under normal conditions and after trauma, but that addition of IGF-1/IGFBP-2 does not have a positive additive effect.

The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion.

DOCK proteins are guanine nucleotide exchange factors for Rac and Cdc42 GTPases. DOCK1 is the founding member of the family and acts downstream of integrins via the canonical Crk-p130Cas complex to activate Rac GTPases in numerous contexts. In contrast, DOCK5, which possesses the greatest similarity to DOCK1, remains sparingly studied. Here we establish that DOCK5 has a non-redundant role in regulating motile and invasive capacities of epithelial cells. DOCK1 is constitutively associated with sites of integrin attachment termed focal adhesions (FAs). In contrast, we demonstrate that DOCK5 recruitment to FAs in Hela cells is restricted by GIT2, an established regulator of FA signaling. We determine that GIT2 is targeted to FAs in response to Rho-ROCK signaling and actomyosin contractility. Accordingly, inhibition of ROCK activity or MLC function promotes enrichment of DOCK5 in membrane protrusions and nascent cell-substratum adhesions. We further demonstrate that GIT2 inhibits the interaction of DOCK5 with Crk. Moreover, we show that depletion of GIT2 promotes DOCK5-dependent activation of the Crk-p130Cas signaling cascade to promote Rac1-mediated lamellipodial protrusion and FA turnover. The antagonism between GIT2 and DOCK5 extends to non-transformed MCF10A mammary epithelial cells, with DOCK5 'dialing-up' and GIT2 'dialing-down' invasiveness. Finally, we determine that DOCK5 inhibition attenuates invasion and metastasis of MDA-MB-231 cells and prolongs life span of mice injected with these cells. Collectively, our work identifies DOCK5 as a key regulator of epithelial invasion and metastasis, and demonstrates that suppression of DOCK5 by GIT2 represents a previously unappreciated mechanism for coordination of Rho and Rac GTPases.

Identification and characterization of an inner ear-expressed human melanoma inhibitory activity (MIA)-like gene (MIAL) with a frequent polymorphism that abolishes translation.

To discover new cochlea-specific genes as candidate genes for nonsyndromic hearing impairment, we searched in The Institute of Genome Research database for expressed sequence tags isolated from the cochlea only. This led to the cloning and characterization of a human gene named melanoma inhibitory activity-like (MIAL; HGMW-approved symbol OTOR alias MIAL) gene. In situ hybridization revealed MIAL expression in a cell layer beneath the sensory epithelium of cochlea and vestibule of human fetal inner ear. No other human tissue, except fetal brain, showed expression of MIAL when analyzed by in situ hybridization or reverse transcription-polymerase chain reaction. The cDNA of the mouse homologue was also cloned and mapped about 80 cM from the top of mouse chromosome 2. In mouse, Mial was also expressed in the cochlea and the vestibule of the inner ear, as well as in brain, eye, limb, and ovary. Expression in mammalian cell cultures showed that MIAL is translated as an approximately 15-kDa polypeptide that is assembled into a covalently linked homodimer, modified by sulfation, and secreted from the cells via the Golgi apparatus. In the human MIAL gene, a frequent polymorphism was discovered in the translation initiation codon (ACG instead of ATG). Of 505 individuals, 48 (9.5%) were ATG/ACG heterozygous and 1 (0.2%) was homozygous for ACG. No MIAL protein was synthesized in cells transfected with cDNA of the ACG allele. The inner ear-restricted expression pattern and the existence of an inactive allele suggest that MIAL may contribute to inner-ear dysfunction in humans.

A phosphoserine-regulated docking site in the protein kinase RSK2 that recruits and activates PDK1.

The 90 kDa ribosomal S6 kinase-2 (RSK2) is a growth factor-stimulated protein kinase with two kinase domains. The C-terminal kinase of RSK2 is activated by ERK-type MAP kinases, leading to autophosphorylation of RSK2 at Ser386 in a hydrophobic motif. The N-terminal kinase is activated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) through phosphorylation of Ser227, and phosphorylates the substrates of RSK. Here, we identify Ser386 in the hydrophobic motif of RSK2 as a phosphorylation-dependent docking site and activator of PDK1. Treatment of cells with growth factor induced recruitment of PDK1 to the Ser386-phosphorylated hydrophobic motif and phosphorylation of RSK2 at Ser227. A RSK2-S386K mutant showed no interaction with PDK1 or phosphorylation at Ser227. Interaction with Ser386-phosphorylated RSK2 induced autophosphorylation of PDK1. Addition of a synthetic phosphoSer386 peptide (RSK2(373-396)) increased PDK1 activity 6-fold in vitro. Finally, mutants of RSK2 and MSK1, a RSK-related kinase, with increased affinity for PDK1, were constitutively active in vivo and phosphorylated histone H3. Our results suggest a novel regulatory mechanism based on phosphoserine-mediated recruitment of PDK1 to RSK2, leading to coordinated phosphorylation and activation of PDK1 and RSK2.

90-kDa ribosomal S6 kinase is phosphorylated and activated by 3-phosphoinositide-dependent protein kinase-1.

90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation of the linker region at Ser(369), catalyzed by extracellular signal-regulated kinase (ERK), and at Ser(386), catalyzed by the C-terminal kinase, after its activation by ERK. In addition, the N-terminal kinase must be phosphorylated at Ser(227) in the activation loop by an as yet unidentified kinase. Here, we show that the isolated N-terminal kinase of RSK2 (amino acids 1-360) is phosphorylated at Ser(227) by PDK1, a constitutively active kinase, leading to 100-fold stimulation of kinase activity. In COS7 cells, ectopic PDK1 induced the phosphorylation of full-length RSK2 at Ser(227) and Ser(386), without involvement of ERK, leading to partial activation of RSK2. Similarly, two other members of the RSK family, RSK1 and RSK3, were partially activated by PDK1 in COS7 cells. Finally, our data indicate that full activation of RSK2 by growth factor requires the cooperation of ERK and PDK1 through phosphorylation of Ser(227), Ser(369), and Ser(386). Our study extend recent findings which implicate PDK1 in the activation of protein kinases B and C and p70(S6K), suggesting that PDK1 controls several major growth factor-activated signal transduction pathways.

Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction.

Extracellular signals activate mitogen-activated protein kinase (MAPK) cascades to execute complex cellular programs, like proliferation, differentiation and apoptosis. In mammalian cells, three MAPK families have been characterized: extracellular signal-regulated kinase (ERK), which is activated by growth factors, peptide hormones and neurotransmitters, and Jun kinase (JNK) and p38 MAPK, which are activated by cellular stress stimulus as well as growth factors. This review describes the family of 90 kDa ribosomal S6 kinases (RSK; also known as p90rsk or MAPK-activated protein kinase-1, MAPKAP-K1), which were among the first substrates of ERK to be discovered and which has proven to be a ubiquitous and versatile mediator of ERK signal transduction. RSK is composed of two functional kinase domains that are activated in a sequential manner by a series of phosphorylations. Recently, a family of RSK-related kinases that are activated by ERK as well as p38 MAPK were discovered and named mitogen- and stress-activated protein kinases (MSK). A number of cellular functions of RSK have been proposed. (1) Regulation of gene expression via association and phosphorylation of transcriptional regulators including c-Fos, estrogen receptor, NFkappaB/IkappaB alpha, cAMP-response element-binding protein (CREB) and CREB-binding protein; (2) RSK is implicated in cell cycle regulation in Xenopus laevis oocytes by inactivation of the Myt1 protein kinase leading to activation of the cyclin-dependent kinase p34cdc2; (3) RSK may regulate protein synthesis by phosphorylation of polyribosomal proteins and glycogen synthase kinase-3; and (4) RSK phosphorylates the Ras GTP/GDP-exchange factor, Sos leading to feedback inhibition of the Ras-ERK pathway.

Transforming growth factor-beta, but not ciliary neurotrophic factor, inhibits DNA synthesis of adrenal medullary cells in vitro.

Transforming growth factor-betas are members of a superfamily of multifunctional cytokines regulating cell growth and differentiation. Their functions in neural and endocrine cells are not well understood. We show here that transforming growth factor-betas are synthesized, stored and released by the neuroendocrine chromaffin cells, which also express the transforming growth factor-beta receptor type II. In contrast to the developmentally related sympathetic neurons, chromaffin cells continue to proliferate throughout postnatal life. Using 5-bromo-2'-deoxyuridine pulse labeling and tyrosine hydroxylase immunocytochemistry as a marker for young postnatal rat chromaffin cells, we show that treatment with fibroblast growth factor-2 (1 nM) and insulin-like growth factor-II (10 nM) increased the fraction of 5-bromo-2'-deoxyuridine-labeled nuclei from 1% to about 40% of the cells in the absence of serum. In the presence of fibroblast growth factor-2 and insulin-like growth factor-II, transforming growth factor-beta1 (0.08 nM) reduced 5-bromo-2'-deoxyuridine labeling by about 50%, without interfering with chromaffin cell survival or death. Doses lower and higher than 0.08 nM were less effective. Similar effects were seen with transforming growth factor-beta3. In contrast to transforming growth factor-beta, ciliary neurotrophic factor, which inhibits proliferation of sympathetic progenitor cells, was not effective on rat chromaffin cells from postnatal day 6. Glucocorticoids also suppress DNA synthesis in fibroblast growth factor-2/insulin-like growth factor-II-treated chromaffin cells. This effect was not mediated by chromaffin cell-derived transforming growth factor-beta, as shown by addition of neutralizing antibodies. We conclude that one function of adrenal medullary transforming growth factor-beta may be to act as a negative regulator of chromaffin cell division.

Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting beta-cell line, INS-1.

The signaling pathways whereby glucose and hormonal secretagogues regulate insulin-secretory function, gene transcription, and proliferation of pancreatic beta-cells are not well defined. We show that in the glucose-responsive beta-cell line INS-1, major secretagogue-stimulated signaling pathways converge to activate 44-kDa mitogen-activated protein (MAP) kinase. Thus, glucose-induced insulin secretion was found to be associated with a small stimulatory effect on 44-kDa MAP kinase, which was synergistically enhanced by increased levels of intracellular cAMP and by the hormonal secretagogues glucagon-like peptide-1 and pituitary adenylate cyclase-activating polypeptide. Activation of 44-kDa MAP kinase by glucose was dependent on Ca2+ influx and may in part be mediated by MEK-1, a MAP kinase kinase. Stimulation of Ca2+ influx by KCl was in itself sufficient to activate 44-kDa MAP kinase and MEK-1. Phorbol ester, an activator of protein kinase C, stimulated 44-kDa MAP kinase by both Ca(2+)-dependent and -independent pathways. Nerve growth factor, independently of changes in cytosolic Ca2+, efficiently stimulated 44-kDa MAP kinase without causing insulin release, indicating that activation of this kinase is not sufficient for secretion. In the presence of glucose, however, nerve growth factor potentiated insulin secretion. In INS-1 cells, activation of 44-kDa MAP kinase was partially correlated with the induction of early response genes junB, nur77, and zif268 but not with stimulation of DNA synthesis. Our findings suggest a role of 44-kDa MAP kinase in mediating some of the pleiotropic actions of secretagogues on the pancreatic beta-cell.

Neuronal localization of pituitary adenylate cyclase-activating polypeptide 38 in the adrenal medulla and growth-inhibitory effect on chromaffin cells.

The chromaffin cells of the adult rat adrenal medulla are essentially growth arrested in situ, but can proliferate in vitro, suggesting the existence of growth inhibitory factors in the adrenal gland. We have investigated whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) could be involved in the growth arrest of adrenal chromaffin cells. In adult rat adrenal gland, PACAP38 was detected by radioimmunoassay and high-performance liquid chromatography and its concentration in the medulla was estimated as 24 nmol/kg wet tissue. Immunohistochemistry of the neonatal and adult rat adrenal medulla showed PACAP38 immunoreactivity in a widely distributed network of delicate nerve fibers surrounding the chromaffin cells. In a primary culture system, PACAP38 inhibited growth factor-stimulated DNA synthesis by 90% in neonatal and adult rat chromaffin cells with half-maximal inhibition at 4 and 0.5 nM, respectively, as demonstrated by bromodeoxyuridine pulse-labeling and immunocytochemical staining of cell nuclei. In comparison, corticosterone inhibited neonatal and adult chromaffin cell proliferation by 70% and 95%, respectively, with half-maximal effect at 100 nM. In neonatal chromaffin cells, 100 nM PACAP38 and 1 microM corticosterone added together abolished proliferation completely (99.8% inhibition). Finally, PACAP38 increased cell survival but showed little neurite-promoting activity in the chromaffin cells. Our data suggest that neurally derived PACAP38, in conjunction with glucocorticoids, may override growth factor mitogenic signals, leading to the postmitotic state of chromaffin cells in the adult adrenal medulla.

Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP.

In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the role of B-Raf in this process. We observed that NGF, PMA and cAMP induce the phosphorylation of B-Raf as well as an upward shift in its electrophoretic mobility. We show that B-Raf is activated following NGF and PMA treatment of PC12 cells, and that it can phosphorylate and activate MEK-1. However, cAMP inhibits B-Raf autokinase activity as well as its ability to phosphorylate and activate MEK-1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf binds to p21ras, but more important, this binding to p21ras is virtually abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf hampers its interaction with p21ras, which is responsible for the PKA-mediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP kinase through the activation of an unidentified MEK kinase and/or the inhibition of a MEK phosphatase.

Insulin-like growth factors act synergistically with basic fibroblast growth factor and nerve growth factor to promote chromaffin cell proliferation.

We have investigated the effects of insulin-like growth factors (IGFs), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF) on DNA synthesis in cultured chromaffin cells from fetal, neonatal, and adult rats by using 5-bromo-2'-deoxyuridine (BrdUrd) pulse labeling for 24 or 48 h and immunocytochemical staining of cell nuclei. After 6 days in culture in the absence of growth factors, nuclear BrdUrd incorporation was detected in 30% of fetal chromaffin cells, 1.5% of neonatal cells, and 0.1% of adult cells. Addition of 10 nM IGF-I or IGF-II increased the fraction of BrdUrd-labeled nuclei to 50% of fetal, 20% of neonatal, and 2% of adult chromaffin cells. The ED50 value of IGF-I- and IGF-II-stimulated BrdUrd labeling in neonatal chromaffin cells was 0.3 nM and 0.8 nM, respectively. In neonatal and adult chromaffin cells, addition of 1 nM bFGF or 2 nM NGF stimulated nuclear BrdUrd incorporation to approximately the same level as 10 nM IGF-I or IGF-II. However, the response to bFGF or NGF in combination with either IGF-I or IGF-II was more than additive, indicating that the combined effect of the IGFs and bFGF or NGF is synergistic. The degree of synergism was 2- to 4-fold in neonatal chromaffin cells and 10- to 20-fold in adult chromaffin cells compared with the effect of each growth factor alone. In contrast, the action of bFGF and NGF added together in the absence of IGFs was not synergistic or additive. IGF-II acted also as a survival factor on neonatal chromaffin cells and the cell survival was further improved when bFGF or NGF was added together with IGF-II. In conclusion, we propose that IGF-I and IGF-II act in synergy with bFGF and NGF to stimulate proliferation and survival of chromaffin cells during neonatal growth and adult maintenance of the adrenal medulla. Our findings may have implications for improving the survival of chromaffin cell implants in diseased human brain.

Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells.

Mitogen-activated protein (MAP) kinases are activated in response to a large variety of extracellular signals, including growth factors, hormones, and neurotransmitters, which activate distinct intracellular signaling pathways. Their activation by the cAMP-dependent pathway, however, has not been reported. In rat pheochromocytoma PC12 cells, we demonstrate here a stimulation of the MAP kinase isozyme extracellular signal-regulated kinase 1 (ERK1) following elevation of intracellular cAMP after exposure of the cells to isobutylmethylxanthine, cholera toxin, forskolin, or cAMP-analogues. cAMP acted synergistically with phorbol ester, an activator of protein kinase C, in the stimulation of ERK1. In accordance with this observation, the peptide neurotransmitter pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), which stimulates cAMP production as well as phosphatidylinositol breakdown in PC12 cells, was an efficient activator of ERK1. In combination with various growth factors, cAMP acted in a more than additive manner on ERK1 activity. Elevation of intracellular cAMP increased in vivo 32P-labeling of ERK1, suggesting that cAMP stimulated ERK1 by activating MAP kinase kinase, an immediate upstream activator of ERK1 in the MAP kinase cascade. Supporting this view, forskolin and a cAMP analogue were found to increase the activity of MAP kinase kinase in PC12 cells, alone as well as in combination with phorbol ester. PACAP38 also stimulated in vivo 32P-labeling of ERK1 and MAP kinase kinase activity. Finally, cAMP or PACAP38 increased by 3-fold nerve growth factor-stimulated neurite formation in PC12 cells, which may be correlated with the potentiating effect of these agents on nerve growth factor-stimulated ERK1 activity.