Human acid alpha-glucosidase from rabbit milk has therapeutic effect in mice with glycogen storage disease type II.

Pompe's disease or glycogen storage disease type II (GSDII) belongs to the family of inherited lysosomal storage diseases. The underlying deficiency of acid alpha-glucosidase leads in different degrees of severity to glycogen storage in heart, skeletal and smooth muscle. There is currently no treatment for this fatal disease, but the applicability of enzyme replacement therapy is under investigation. For this purpose, recombinant human acid alpha-glucosidase has been produced on an industrial scale in the milk of transgenic rabbits. In this paper we demonstrate the therapeutic effect of this enzyme in our knockout mouse model of GSDII. Full correction of acid alpha-glucosidase deficiency was obtained in all tissues except brain after a single dose of i.v. enzyme administration. Weekly enzyme infusions over a period of 6 months resulted in degradation of lysosomal glycogen in heart, skeletal and smooth muscle. The tissue morphology improved substantially despite the advanced state of disease at the start of treatment. The results have led to the start of a Phase II clinical trial of enzyme replacement therapy in patients.

Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho.

Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.

cAMP abrogates the p21ras-mitogen-activated protein kinase pathway in fibroblasts.

The mechanism by which cAMP inhibits growth factor-induced DNA synthesis in fibroblasts is not understood. Here we show that in Rat-1 fibroblasts, cAMP-raising agents inhibit p21ras-mediated mitogen-activated protein (MAP) kinase activation induced by either epidermal growth factor or lysophosphatidic acid. Under the same conditions, however, epidermal growth factor- or lysophosphatidic acid-induced protein tyrosine phosphorylation, Ca2+ mobilization, and activation of Na+/H+ exchange are not attenuated. In ras-transformed Rat-1 cells, 8-bromo-cAMP rapidly deactivates constitutively active MAP kinase without reducing p21ras.GTP levels; long term 8-bromo-cAMP treatment of these cells leads to growth arrest and reversion of the transformed phenotype. These results show that elevation of intracellular cAMP levels abrogates the p21ras MAP kinase pathway at a step downstream of p21ras activation. This finding provides a molecular basis for the growth-inhibitory action of cAMP in normal and transformed fibroblasts.

Protein tyrosine phosphorylation induced by lysophosphatidic acid in Rat-1 fibroblasts. Evidence that phosphorylation of map kinase is mediated by the Gi-p21ras pathway.

Lysophosphatidic acid (LPA) is a platelet-derived phospholipid that serves as a mitogen for fibroblasts. LPA activates its own G protein-coupled receptor(s) leading to stimulation of phospholipase C and inhibition of adenylate cyclase. Furthermore, LPA rapidly activates p21ras through a pertussis toxin-sensitive pathway. In this study, we have examined LPA-induced protein tyrosine phosphorylation in Rat-1 fibroblasts. LPA action was compared with that of endothelin, which is a stronger activator of phospholipase C than LPA but fails to activate p21ras and to stimulate DNA synthesis in these cells. LPA and, more effectively, endothelin rapidly stimulate tyrosine phosphorylation of proteins of 110-130, 95, and 65-75 kDa. The effect of LPA is dose- and time-dependent, being half-maximal at 3-30 nM and peaking after 2-5 min. Among the 110-130-kDa group of phosphotyrosyl proteins is the 125-kDa "focal adhesion kinase" (p125FAK) but not the 120-kDa p21ras GTPase-activating protein. Furthermore, LPA, like epidermal growth factor, causes tyrosine phosphorylation and activation of the p42/p44 mitogen-activated protein (MAP) kinases, paralleling p21ras activation. In contrast, endothelin fails to phosphorylate MAP kinase. Treatment of the cells with pertussis toxin blocks LPA-induced MAP kinase phosphorylation without affecting the other tyrosine phosphorylations. The kinase inhibitor staurosporine (1 microM) blocks LPA-induced, but not epidermal growth factor-induced, activation of p21ras and MAP kinase, consistent with an intermediate protein kinase linking the LPA receptor to p21ras activation. The results support a model in which LPA-induced phosphorylation of MAP kinase is mediated by p21ras, and tyrosine phosphorylation of the other substrates, including p125FAK, is associated with phospholipase C activation.

Gi-mediated activation of the p21ras-mitogen-activated protein kinase pathway by alpha 2-adrenergic receptors expressed in fibroblasts.

The alpha 2-adrenergic receptors are linked to inhibition of adenylylcyclase and, under certain circumstances, to stimulation of phospholipid hydrolysis via pertussis toxin-sensitive G proteins. Here we show that alpha 2-adrenergic receptors can couple to an alternative signaling pathway. When expressed in Rat-1 cells, stimulation of the alpha 2A receptor, which couples to Gi2 and Gi3, causes rapid, transient activation of the protooncogene product p21ras as measured by an increase in the amount of bound GTP. Furthermore, alpha 2A receptor stimulation causes rapid phosphorylation of the p42 mitogen-activated protein (MAP) kinase. Pertussis toxin completely inhibits both p21ras activation and MAP kinase phosphorylation, but both responses appear to be independent of adenylylcyclase inhibition or phospholipase stimulation. Thus, alpha 2-adrenergic receptors can couple to the p21ras-MAP kinase pathway via Gi, which may explain the mitogenic potential of alpha 2 agonists in certain cell types; together with previous results, these findings further suggest that activation of this pivotal signaling pathway may be a common event in the action of Gi-coupled receptors.

Lysophosphatidic acid induces neuronal shape changes via a novel, receptor-mediated signaling pathway: similarity to thrombin action.

Lysophosphatidic acid (LPA) is a mitogenic phospholipid produced by certain activated cells and present in serum. LPA stimulates phospholipase C and inhibits adenylate cyclase in its target cells, apparently by activating a specific G-protein-coupled receptor. Here, we demonstrate that LPA causes transient rounding of N1E-115 and NG108-15 neuronal cells accompanied by growth cone collapse and retraction of neurites. The effect of LPA is concentration dependent, being half-maximal at 10-20 nM, and reversibly blocked by suramin, an LPA receptor antagonist. The morphological response to LPA is indistinguishable from that evoked by thrombin or a thrombin receptor-activating peptide (TRP) (K. Jalink and W. H. Moolenaar, J. Cell Biol., 118: 411-419, 1992); yet, LPA and thrombin appear to act through distinct receptors. LPA-induced shape changes, like those induced by thrombin and TRP, are driven by contraction of the cortical actin cytoskeleton and not attributable to prior phospholipid hydrolysis and Ca2+ mobilization nor to other classic second messenger systems. Instead, LPA- and TRP-induced shape changes are accompanied by a small but significant increase in p60src protein tyrosine kinase activity. Treatment of cells with pervanadate selectively inhibits LPA- and TRP-induced shape changes as well as p60src activation. These results indicate that, in N1E-115 and NG108-15 cells, LPA and TRP trigger neurite retraction and cell rounding through a novel, receptor-mediated signaling pathway, and they suggest that p60src may play a role in this pathway.

Pertussis toxin-sensitive activation of p21ras by G protein-coupled receptor agonists in fibroblasts.

Some agonists of G protein-coupled receptors, such as thrombin and lysophosphatidic acid (LPA), can promote cell proliferation via a pertussis toxin (PTX)-sensitive signaling pathway. While these agonists stimulate phospholipase C and inhibit adenylate cyclase, it appears that other, as-yet-unidentified, effector pathways are required for mitogenesis. Here we report that LPA and a thrombin receptor agonist peptide rapidly activate the protooncogene product p21ras in quiescent fibroblasts. This activation is inhibited by PTX and yet not attributable to known PTX-sensitive G protein pathways, including stimulation of phospholipases, inhibition of adenylate cyclase, or modulation of ion channels. LPA- and peptide-induced p21ras activation is inhibited by the tyrosine kinase inhibitor genistein, at doses that do not affect epidermal growth factor-induced p21ras activation. Thus, a heterotrimeric G protein of the Gi subfamily regulates activation of p21ras by LPA and thrombin, possibly through an intermediary tyrosine kinase. This pathway may critically participate in mitogenic signaling downstream from certain G protein-coupled receptors.

The biologically active phospholipid, lysophosphatidic acid, induces phosphatidylcholine breakdown in fibroblasts via activation of phospholipase D. Comparison with the response to endothelin.

Lysophosphatidic acid (LPA) is a simple phospholipid that possesses hormone- and growth-factor-like properties. LPA initiates its action by inducing GTP-dependent phosphoinositide hydrolysis and inhibiting adenylate cyclase [van Corven, Groenink, Jalink, Eichholtz & Moolenaar (1989) Cell 59, 45-54]. Here we show that LPA stimulates rapid breakdown of phosphatidylcholine (PC) in Rat-1 fibroblasts. LPA-induced PC breakdown occurs through activation of phospholipase D (PLD), as measured by the formation of free choline and phosphatidic acid and by transphosphatidylation in the presence of butan-1-ol. LPA also stimulates generation of diacylglycerol, but there is no detectable formation of phosphocholine, suggesting that a PC-specific phospholipase C (PLC) is not involved. The response to LPA was compared with that to endothelin, a potent inducer of phospholipid hydrolysis but a poor mitogen for Rat-1 cells. Our results indicate that: (1) LPA is less efficient than endothelin in inducing phosphoinositide and PC breakdown; (2) LPA-induced PLD activation is short-lived, levelling off after 2 min, whereas the endothelin-stimulated increase in PLD activity persists for at least 1 h; (3) the effect of LPA on PLD, like that of endothelin, is blocked by long-term pretreatment of the cells with phorbol ester, suggesting that PLD activation occurs through a protein kinase C-dependent mechanism. Furthermore, our results support the notion that there is no simple causal relationship between the degree of agonist-induced phospholipid hydrolysis and the magnitude of the mitogenic response.

Identification of a putative membrane receptor for the bioactive phospholipid, lysophosphatidic acid.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with hormone- and growth factor-like activities. Exogenous LPA stimulates GTP-dependent phosphoinositide hydrolysis and inhibits adenylate cyclase in its target cells, but the site of action of LPA is unknown. We now report the identification by photoaffinity labeling of a putative LPA membrane receptor in various LPA-responsive cell types. A 32P-labeled LPA analogue containing a photoreactive fatty acid, [32P]diazirine-LPA, labels a membrane protein of apparent molecular mass of 38-40 kDa in various cell types, including neuronal cells, brain homogenates, carcinoma cells, leukemic cells and normal fibroblasts. Labeling of the 38-40 kDa protein is competitively inhibited by unlabeled 1-oleoyl-LPA (IC50 approximately 10 nM), but not by other phospholipids. Specific labeling is not detected in rat liver membranes or in human neutrophils, which are physiologically unresponsive to LPA. Suramin, an inhibitor of both early and late events in the action of LPA, completely inhibits the binding of photoreactive LPA. We suggest that the 38-40 kDa protein represents a specific LPA cell surface receptor mediating at least part of the multiple cellular responses to LPA.

Metabolic conversion of the biologically active phospholipid, lysophosphatidic acid, in fibroblasts.

Lysophosphatidic acid (3-sn-lysophosphatidic acid; LPA) can activate cells similar to hormones and growth factors. We have considered the question whether metabolic conversion of LPA taken up by the cell could be of any importance in this activation. Addition of [14C-glycerol]LPA to quiescent Rat-1 fibroblasts resulted in rapid formation of [14C]monoacylglycerol (MG), closely followed by accumulation of [14C]triacylglycerol. Only very little [14C]diacylglycerol and [14C]phosphatidic acid was formed (approx. 100-fold less than MG). MG, when added exogenously to cells, lacks detectable biological activity. The results suggest that LPA itself, rather than one of its metabolites is the biologically active principle.

Mitogenic action of lysophosphatidic acid and phosphatidic acid on fibroblasts. Dependence on acyl-chain length and inhibition by suramin.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with growth-factor-like activities [van Corven, Groenink, Jalink, Eichholtz & Moolenaar (1989) Cell 45, 45-54]. We have examined various structural analogues of LPA for their ability to stimulate DNA synthesis in quiescent fibroblasts. When the acyl-chain length is varied, the rank order of mitogenic potency is: 1-oleoyl LPA congruent to 1-palmitoyl LPA greater than 1-myristoyl LPA greater than 1-lauroyl LPA greater than 1-decanoyl LPA; the last compound shows almost no activity over the concentration range tested (1-100 microM). An ether-linked LPA (1-O-hexadecylglycerol 3-phosphate) has much decreased mitogenic activity as compared with the ester-linked analogue at concentrations less than 25 microM, and becomes cytotoxic at higher concentrations. Hexadecylphosphate, which lacks a glycerol backbone, has negligible activity. On a molar basis, diacyl phosphatidic acid (PA) is about equally potent as the corresponding LPA analogue, showing similar acyl-chain-length dependence; the data argue against the possibility that the mitogenic action of PA is due to contaminating traces of LPA. Although the short-chain analogues of LPA and PA fail to antagonize the action of long-chain (L)PAs, the polyanionic drug suramin inhibits LPA- and PA-induced, DNA synthesis in a reversible and dose-dependent manner, at concentrations [IC50 (concn. giving 50% inhibition) approximately 70 microM] that do not affect epidermal-growth-factor-induced DNA synthesis. Suramin appears to act in the early G0/G1 phase of the cell cycle, blocking immediate responses to LPA such as phosphoinositide hydrolysis. We conclude that both LPA and PA can function as growth-promoting phospholipids, with the fatty acid chain length being a major determinant of mitogenic potency.

Lysophosphatidic acid, but not phosphatidic acid, is a potent Ca2(+)-mobilizing stimulus for fibroblasts. Evidence for an extracellular site of action.

Lysophosphatidic acid (LPA) is a potent mitogen for quiescent fibroblasts. Among the earliest detectable responses to LPA is GTP-dependent phosphoinositide hydrolysis (van Corven, E. J., Groenink, A., Jalink, K., Eichholtz, T., and Moolenaar, W. H. (1989) Cell 59, 45-54). Here we describe the Ca2(+)-mobilizing properties of LPA in human fibroblasts and present evidence suggesting that previously reported Ca2(+)-mobilizing effects of phosphatidic acid are attributable to contamination with LPA. Addition of LPA (1-oleoyl or 1-palmitoyl) to fibroblasts evokes the formation of inositol 1,4,5-trisphosphate accompanied by an immediate but transient rise in [Ca2+]i which originates primarily from intracellular stores. The Ca2+ response is dose-dependent with a half-maximal effect at LPA concentrations as low as 10 ng/ml, far below the reported half-maximal effect for DNA synthesis (5-10 micrograms/ml). LPA-induced Ca2+ release is also observed in various other cell types, both normal and transformed, but not in Jurkat T cells and neutrophils. The Ca2(+)-mobilizing action of LPA is specific, in that 1,2-dioleoyl-phosphatidic acid (when prepared free of LPA contaminants), other lysophospholipids, monoacylglycerol, and free fatty acids have no effect. Furthermore, LPA, unlike lysophosphatidylcholine, does not cause detectable membrane leakiness, even when added at high concentrations (500 micrograms/ml). The LPA-induced Ca2+ signal is blocked completely by tetradecanoylphorbol acetate, but is not affected by prior stimulation of the cells with Ca2(+)-mobilizing agonists such as bradykinin or histamine. In contrast, pretreating the cells with a low dose of LPA desensitizes the Ca2+ response to subsequent addition of higher doses. This homologous desensitization is not inhibited by staurosporine, nor by down-regulating protein kinase C with tetradecanoylphorbol acetate, suggesting independence of functional protein kinase C activity. Addition of La3+ instantaneously blocks inositol phosphate production and Ca2+ mobilization in response to LPA, but not to bradykinin, most likely due to formation of inactive La3(+)-LPA complexes, suggesting that LPA acts at an extracellular site on the plasma membrane to trigger GTP-dependent phosphoinositide breakdown.

Growth factor-like action of lysophosphatidic acid: mitogenic signalling mediated by G proteins.

Several classes of growth factors can be distinguished that act through different signal transduction pathways. One class is constituted by the peptide growth factors that bind to receptors with ligand-dependent protein tyrosine kinase activity. Another class of mitogens activates a phosphoinositide-specific phospholipase C via a receptor-linked G protein. An intriguing member of this class is lysophosphatidic acid (LPA). LPA mitogenicity is not dependent on other mitogens and is blocked by pertussis toxin. LPA evokes at least three separate signalling cascades: (i) activation of a pertussis toxin-insensitive G protein mediating phosphoinositide hydrolysis; (ii) release of arachidonic acid in a GTP-dependent manner, but independent of prior phosphoinositide hydrolysis; and (iii) activation of a pertussis toxin-sensitive Gi protein mediating inhibition of adenylate cyclase. The peptide bradykinin mimics LPA in inducing responses (i) and (ii), but fails to activate Gi and to stimulate DNA synthesis. Our results suggest that the mitogenic action of LPA occurs through Gi or a related pertussis toxin substrate and that, unexpectedly, the phosphoinositide hydrolysis pathway is neither required nor sufficient, by itself, for mitogenesis.

Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins.

Lysophosphatidate (LPA), the simplest natural phospholipid, is highly mitogenic for quiescent fibroblasts. LPA-induced cell proliferation is not dependent on other mitogens and is blocked by pertussis toxin. LPA initiates at least three separate signaling cascades: activation of a pertussis toxin-insensitive G protein mediating phosphoinositide hydrolysis with subsequent Ca2+ mobilization and stimulation of protein kinase C; release of arachidonic acid in a GTP-dependent manner, but independent of prior phosphoinositide hydrolysis; and activation of a pertussis toxin-sensitive Gi protein mediating inhibition of adenylate cyclase. The peptide bradykinin mimics LPA in inducing the first two responses but fails to activate Gi and to stimulate DNA synthesis. Our data suggest that the mitogenic action of LPA occurs through Gi or a related pertussis toxin substrate and that the phosphoinositide hydrolysis-protein kinase C pathway is neither required nor sufficient, by itself, for mitogenesis. The results further suggest that LPA or LPA-like phospholipids may have a novel role in G protein-mediated signal transduction.

Calcium homeostasis of epithelial cells.

1. Cytosolic free Ca2+ is an important regulator of ion transport processes in epithelial cells. 2. Free Ca2+ concentration is regulated by a concerted action of Ca2+ transport systems in plasma membranes and intracellular organelles. 3. These transport systems were studied in intestinal and renal cortical cells with emphasis on the transport capacities and Ca2+ affinities. 4. Ca2+ accumulation by permeabilized cells was compared to Ca2+ uptake by isolated organelles and membrane fractions. 5. Effects induced by cell or organelle isolation methods and the influence of temperature and pH on Ca2+ transport capacities were studied.