Peter Gierschik

SDF-1α/CXCR4 Axis Is Instrumental in Neointimal Hyperplasia and Recruitment of Smooth Muscle Progenitor Cells

Recent evidence infers a contribution of smooth muscle cell (SMC) progenitors and stromal cell-derived factor (SDF)-1&agr; to neointima formation after arterial injury. Inhibition of plaque area and SMC content in apolipoprotein E-deficient mice repopulated with LacZ+ or CXCR4−/− BM or lentiviral transfer of an antagonist reveals a crucial involvement of local SDF-1&agr; and its receptor CXCR4 in neointimal hyperplasia via recruitment of BM-derived SMC progenitors. After arterial injury, SDF-1&; expression in medial SMCs is preceded by apoptosis and inhibited by blocking caspase-dependent apoptosis. SDF-1&agr; binds to platelets at the site of injury, triggers CXCR4- and P-selectin-dependent arrest of progenitor cells on injured arteries or matrix-adherent platelets, preferentially mobilizes and recruits c-kit−/platelet–derived growth factor receptor (PDGFR)-&bgr;+/lineage−/sca-1+ progenitors for neointimal SMCs without being required for their differentiation. Hence, the SDF-1&agr;/CXCR4 axis is pivotal for vascular remodeling by recruiting a subset of SMC progenitors in response to apoptosis and in concert with platelets, epitomizing its importance for tissue repair and identifying a prime target to limit lesion development.

The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI.

Rho family-specific guanine nucleotide dissociation inhibitors (RhoGDIs) decrease the rate of nucleotide dissociation and release Rho proteins such as RhoA, Rac and Cdc42 from membranes, forming tight complexes that shuttle between cytosol and membrane compartments. We have solved the crystal structure of a complex between the RhoGDI homolog LyGDI and GDP-bound Rac2, which are abundant in leukocytes, representing the cytosolic, resting pool of Rho species to be activated by extracellular signals. The N-terminal domain of LyGDI (LyN), which has been reported to be flexible in isolated RhoGDIs, becomes ordered upon complex formation and contributes more than 60% to the interface area. The structure is consistent with the C-terminus of Rac2 binding to a hydrophobic cavity previously proposed as isoprenyl binding site. An inner segment of LyN forms a helical hairpin that contacts mainly the switch regions of Rac2. The architecture of the complex interface suggests a mechanism for the inhibition of guanine nucleotide dissociation that is based on the stabilization of the magnesium (Mg2+) ion in the nucleotide binding pocket.

Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1.

Neutrophils contain a soluble guanine‐nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C‐β2 (PLCβ2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCβ2 is stimulated by guanosine 5′‐O‐(3‐thiotriphosphate) (GTP[S])‐activated Cdc42Hs×LyGDI, but not by RhoA×LyGDI. Stimulation of PLCβ2, which was also observed for GTP[S]‐activated recombinant Rac1, was independent of LyGDI, but required C‐terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCβ2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein–protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCβ2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCβ2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low‐molecular‐mass GTP‐binding proteins.

Clinical implications of guanine nucleotide-binding proteins as receptor-effector couplers.

IT has long been known that some cellular proteins bind guanine nucleotides such as guanosine tri-phosphate (GTP) with high affinity. These binding proteins are called G proteins and have been link...

A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice

Constitutively active Ras proteins, their regulatory components, and overexpressed tyrosine kinase receptors that activate Ras, are frequently associated with cell transformation in human tumors. This suggests that functional Ras antagonists may have anti-tumor activity. Studies in rodent fibroblasts have shown that S-trans, transfarnesylthiosalicylic acid (FTS) acts as a rather specific nontoxic Ras antagonist, dislodging Ras from its membrane anchorage domains and accelerating its degradation. FTS is not a farnesyltransferase inhibitor, and does not affect Ras maturation. Here we demonstrate that FTS also acts as a functional Ras antagonist in human pancreatic cell lines that express activated K-Ras (Panc-1 and MiaPaCa-2). In Panc-1 cells, FTS at a concentration of 25 – 100 μM reduced the amount of Ras in a dose-dependent manner and interfered with serum-dependent and epidermal growth factor-stimulated ERK activation, thus inhibiting both anchorage-dependent and anchorage-independent growth of Panc-1 cells in vitro. FTS also inhibited tumor growth in Panc-1 xenografted nude mice, apparently without systemic toxicity. Daily FTS treatment (5 mg/kg intraperitoneally) in mice with tumors (mean volume 0.07 cm3) markedly decreased tumor growth (after treatment for 18 days, tumor volume had increased by only 23±30-fold in the FTS-treated group and by 127±66-fold in controls). These findings suggest that FTS represents a new class of functional Ras antagonists with potential therapeutic value.

Growth factor-dependent activation of the Ras-Raf-MEK-MAPK pathway in the human pancreatic carcinoma cell line PANC-1 carrying activated K-ras: implications for cell proliferation and cell migration.

Human ductal adenocarcinoma of the pancreas frequently carry activating point mutations in the K-ras protooncogene. We have analysed the activity of the Ras-Raf-MEK-MAPK cascade in the human pancreatic carcinoma cell line PANC-1 carrying an activating K-ras mutation. Serum-starved cells and cells grown in medium with serum did not show constitutively activated c-Raf, MEK-1, or p42 MAPK. Stimulation of cells with epidermal growth factor (EGF) or fetal calf serum (FCS) resulted in activation of N-Ras, but not K-Ras, as well as activation of c-Raf, MEK-1, and p42 MAPK. Preincubation of serum-starved cells with MEK-1 inhibitor PD98059 abolished EGF- and FCS-induced MAPK activation, identifying MEK as the upstream activator of MAPK. PANC-1 cells exhibited marked serum-dependence of anchorage-dependent and -independent cell growth as well as cell migration. EGF, alone or in combination with insulin and transferrin, did not induce cell proliferation of serum-starved PANC-1 cells, indicating that activation of MAPK alone was not sufficient to induce cell proliferation. FCS-induced DNA synthesis was inhibited by 40% by the MEK-1 inhibitor. On the other hand, treatment with either FCS or EGF alone resulted in marked, MEK-dependent increase of directed cell migration. Collectively, our results show that the activating K-ras mutation in PANC-1 cells does not result in constitutively increased Raf-MEK-MAPK signaling. Signal transduction via the Ras-Raf-MEK-MAPK cascade is maintained in these cells and is required for growth factor-induced cell proliferation and directed cell migration.

TGFβ1 represses proliferation of pancreatic carcinoma cells which correlates with Smad4-independent inhibition of ERK activation

Transforming growth factor beta (TGFβ) is a tumor suppressor acting as inhibitor of cell cycle progression of epithelial cells. We show that treatment of the pancreatic carcinoma cell lines PANC-1 and BxPC-3 with TGFβ1 inhibits both growth factor-induced activation of the extracellular signal-regulated kinase 2 (ERK2) and translocation of the kinase to the nucleus. TGFβ1 causes a concentration-dependent reduction of cell proliferation in both cell lines. By measuring ERK activation, we can show that TGFβ1 is able to repress ERK activation induced by mitogenic stimuli such as EGF. This inhibitory effect of TGFβ1 is not mediated by suppression of Ras or c-Raf-1 activation, but mediated by TGFβ1-induced activation of a serine-threonine phosphatase, as demonstrated by inhibition of phosphatases by treatment with okadaic acid. Results obtained in the Smad4-deficient pancreatic carcinoma cell line BxPC-3, demonstrate that TGFβ1-induced growth inhibition is mediated by a Smad4-independent prevention of ERK2 activation. In contrast to the effects of TGFβ1 on epithelial cells, mesenchymal NIH3T3 fibroblasts exhibit elevated ERK2 activation and increased cell proliferation in response to TGFβ1 treatment. Smad4-independent phosphatase-mediated inhibition of mitogen-activated ERK2 represents a novel effector pathway contributing to suppression of epithelial pancreatic carcinoma cell proliferation by TGFβ1, in addition to the well-known Smad-induced tumor suppressor activity of TGFβ.

A New Missense Mutation in the Leptin Gene Causes Mild Obesity and Hypogonadism without Affecting T Cell Responsiveness

OBJECTIVE Leptin, a protein product of adipocytes, plays a critical role in the regulation of body weight, immune function, pubertal development, and fertility. So far, only three homozygous mutations in the leptin gene in a total of 13 individuals have been found leading to a phenotype of extreme obesity with marked hyperphagia and impaired immune function. DESIGN Serum leptin was measured by ELISA. The leptin gene (OB) was sequenced in patient DNA. The effect of the identified novel mutation was assessed using HEK293 cells. RESULTS We describe a 14-yr-old child of nonobese Austrian parents without known consanguinity. She had a body mass index of 31.5 kg/m(2) (+2.46 SD score) and undetectable leptin serum levels. Sequencing of the leptin gene revealed a hitherto unknown homozygous transition (TTA to TCA) in exon 3 of the LEP gene resulting in a L72S replacement in the leptin protein. RT-PCR, Western blot, and immunohistochemical analysis indicated that the mutant leptin was expressed in the patients adipose tissue but retained within the cell. Using a heterologous cell system, we confirmed this finding and demonstrated that the side chain of Leu72 is crucial for intracellular leptin trafficking. Our patient showed signs of a hypogonadotropic hypogonadism. However, in contrast to the literature, she showed only mild obesity and a normal T cell responsiveness. CONCLUSIONS These findings shed a new light on the clinical consequences of leptin deficiency. Congenital leptin deficiency should be considered possible in pediatric patients with mild obesity even if parents are lean and unrelated.

Mice deficient in the chemokine receptor CXCR4 exhibit impaired limb innervation and myogenesis.

The chemokine CXCL12/SDF-1 and its receptor CXCR4 regulate the development and the function of the hematopoietic system and control morphogenesis of distinct brain areas. Here, we demonstrate that inactivation of CXCR4 results in a massive loss of spinal cord motoneurons and dorsal root ganglion neurons and, subsequently, in a reduced innervation of the developing mouse fore- and hindlimbs. However, only the death of sensory neurons seems to be a direct consequence of receptor inactivation as suggested by the observations that DRG neurons, but not motoneurons, of wild-type animals express CXCR4 and respond to CXCL12 with an increase in cell survival. In contrast, the increased death of motoneurons in CXCR4-deficient animals seems to result from impaired limb myogenesis and a subsequent loss of muscle-derived neurotrophic support. In summary, our findings unravel a previously unrecognized complex role of CXCL12/CXCR4 in the control of limb neuromuscular development.

Interleukin-6 and cAMP Induce Stromal Cell-derived Factor-1 Chemotaxis in Astroglia by Up-regulating CXCR4 Cell Surface Expression IMPLICATIONS FOR BRAIN INFLAMMATION

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 control the migration of neurons and microglial cells in the central nervous system. Although functional CXCR4 is also expressed by astroglia, recent studies have failed to observe a chemotactic response of these cells to SDF-1. Here, we demonstrate that SDF-1-dependent chemotaxis can be induced by treating cultured cortical astroglia with either dibutyryl cyclic AMP (dbcAMP; 10−4 m) or interleukin-6 (IL-6; 10 ng/ml). Flow cytometric analysis revealed that both the dbcAMP- and IL-6-induced onset of SDF-1-dependent chemotaxis of astroglia are due to the increased cell surface expression of CXCR4. In addition, dbcAMP and IL-6 also increased CXCR4 transcript levels, further suggesting that both treatments primarily affect CXCR4 surface expression in astroglia by stimulation of gene expression. Moreover, unlike the case with IL-6 and dbcAMP, which allowed for an optimal chemotactic response to SDF-1 only after 48 h, a similar chemotactic response, associated with an increase in CXCR4 cell surface expression, already occurred after 24 h when astroglial cultures were maintained with medium conditioned by IL-6- or dbcAMP-pretreated astrocytes, indicating that the stimulatory effects of IL-6 and cAMP on CXCR4 cell surface expression involve a secondary mechanism. The findings that elevated extracellular levels of IL-6 or factors positively coupled to cAMP result in increased CXCR4 cell surface expression levels and subsequent SDF-1-dependent chemotaxis in central nervous system astrocytes point to a crucial role of this chemokine during reactive gliosis and human immunodeficiency virus-mediated dementia.