Endostatin regulates branching morphogenesis of renal epithelial cells and ureteric bud.

Endostatin (ES) inhibits endothelial cell migration and has been found to bind to glypicans (Gpcs) on both endothelial cells and renal epithelial cells. We examined the possibility that ES might regulate epithelial cell morphogenesis. The addition of ES to cultured epithelial cells causes an inhibition of both hepatocyte growth factor- and epidermal growth factor-dependent process formation and migration. In contrast, ES does not inhibit epidermal growth factor-dependent morphogenesis in renal epithelial cells derived from Gpc-3 -/mice, whereas expression of Gpc-1 in these cells reconstitutes ES responsiveness. Gpc-3 -/mice have been shown to display enhanced ureteric bud (UB) branching early in development, and cultured UB cells release ES into the media, suggesting that ES binding to Gpcs may regulate UB branching. The addition of ES inhibits branching of the explanted UB, whereas a neutralizing Ab to ES enhances UB outgrowth and branching. Thus, local expression of ES at the tips of the UB may play a role in the regulation of UB arborization.

Cell surface glypicans are low-affinity endostatin receptors.

Endostatin, a collagen XVIII fragment, is a potent anti-angiogenic protein. We sought to identify its endothelial cell surface receptor(s). Alkaline phosphatase- tagged endostatin bound endothelial cells revealing two binding affinities. Expression cloning identified glypican, a cell surface proteoglycan as the lower-affinity receptor. Biochemical and genetic studies indicated that glypicans' heparan sulfate glycosaminoglycans were critical for endostatin binding. Furthermore, endostatin selected a specific octasulfated hexasaccharide from a sequence in heparin. We have also demonstrated a role for endostatin in renal tubular cell branching morphogenesis and show that glypicans serve as low-affinity receptors for endostatin in these cells, as in endothelial cells. Finally, antisense experiments suggest the critical importance of glypicans in mediating endostatin activities.

Differential MAPK pathways utilized for HGF- and EGF-dependent renal epithelial morphogenesis.

Cells derived from the inner medullary collecting duct undergo in vitro branching tubulogenesis to both the c-met receptor ligand hepatocyte growth factor (HGF) as well as epidermal growth factor (EGF) receptor ligands. In contrast, many other cultured renal epithelial cells respond in this manner only to HGF, suggesting that these two receptors may use independent signaling pathways during morphogenesis. We have therefore compared the signaling pathways for mIMCD-3 cell morphogenesis in response to EGF and HGF. Inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway with the mitogen-activated protein kinase kinase (MKK1) inhibitor PD98059 (50 microm) markedly inhibits HGF-induced cell migration with only partial inhibition of EGF-induced cell motility. Similarly, HGF-dependent, but not EGF-dependent, branching morphogenesis was more greatly inhibited by the MKK1 inhibitor. Examination of EGF-stimulated cells demonstrated that extracellular-regulated kinase 5 (ERK5) was activated in response to EGF but not HGF, and that activation of ERK5 was only 60% inhibited by 50 microm PD98059. In contrast, the MKK inhibitor U0126 markedly inhibited both ERK1/2 and ERK5 activation and completely prevented HGF- and EGF-dependent migration and branching process formation. Expression of dominant negative ERK5 (dnBMK1) likewise inhibited EGF-dependent branching process formation, but did not affect HGF-dependent branching process formation. Our results indicate that activation of the ERK1/ERK2 signaling pathway is critical for HGF-induced cell motility/morphogenesis in mIMCD-3 cells, whereas ERK5 appears to be required for EGF-dependent morphogenesis.

Alternate splicing in human Na+-MI cotransporter gene yields differentially regulated transport isoforms.

myo-Inositol is a ubiquitous intracellular organic osmolyte and phosphoinositide precursor maintained at millimolar intracellular concentrations through the action of membrane-associated Na+-myo-inositol cotransporters (SMIT). Functional cloning and expression of a canine SMIT cDNA, which conferred SMIT activity in Xenopus oocytes, predicted a 718-amino acid peptide homologous to the Na+-glucose cotransporter with a potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites. A consistent approximately 1.0- to 13.5-kb array of transcripts hybridizing with this cDNA are osmotically induced in a variety of mammalian cells and species, yet SMIT activity appears to vary among different tissues and species. An open reading frame on human chromosome 21 (SLC5A3) homologous to that of the canine cDNA (96.5%) is thought to comprise an intronless human SMIT gene. Recently, this laboratory ascribed multiply sized, osmotically induced SMIT transcripts in human retinal pigment epithelial cells to the alternate utilization of several 3'-untranslated SMIT exons. This article describes an alternate splice donor site within the coding region that extends the open reading frame into the otherwise untranslated 3' exons, potentially generating novel SMIT isoforms. In these isoforms, the last putative transmembrane domain is replaced with intracellular carboxy termini containing a novel potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites, and this could explain the heterogeneity in the regulation and structure of the SMIT.

Protein kinase and Ca2+ modulation of myo-inositol transport in cultured retinal pigment epithelial cells.

The acute regulation of inwardly directed Na(+)-myo-inositol (MI) cotransporter activity and basal and volume-sensitive MI efflux by protein kinases C (PKC) and A (PKA), cytosolic Ca2+, and phosphoinositide (PI) turnover were characterized in cultured human retinal pigment epithelial cells using 2-[3H]MI and liquid scintillation spectrometry. Kinetic analysis revealed two distinct Na(+)-MI cotransporter components differing in apparent Michaelis constant and maximal velocity. Composite Na(+)-MI cotransport activity was stimulated by PKA activation, the muscarinic agonist carbachol, and the Ca2+ ionophore A-23187 and was inhibited by PKC activation. PKC activation also increased MI efflux, but only the volume-sensitive component, whereas PKA activation increased both basal and volume-sensitive MI efflux. These studies implicate PKC as a negative modulator of MI content through Na(+)-MI cotransport inhibition and potentiation of volume-sensitive MI efflux. PKA is a positive modulator of both Na(+)-MI cotransport and basal and volume-sensitive MI efflux. Cytosolic Ca2+ release through receptor-mediated PI hydrolysis may facilitate Na(+)-MI cotransport activity.

Effect of sorbinil and ascorbic acid on myo-inositol transport in cultured rat Schwann cells exposed to elevated extracellular glucose.

The effect of long-term (2 weeks) exposure to 0-50 mM glucose and 0-1 mM sorbitol on myo-inositol metabolism was studied in cultured rat Schwann cells. Experiments were carried out to determine the effect of sorbinil and ascorbic acid on myo-inositol uptake in rat Schwann cells cultured in the presence of increased extracellular glucose or sorbitol. myo-Inositol uptake and its incorporation into phospholipids decreased significantly when cells were grown in > or = 30 mM glucose for a period of 2 weeks. This inhibitory effect was partly blocked by sorbinil, an aldose reductase inhibitor, in a dose-dependent fashion. Significant prevention was achieved with 0.5 and 1 mM sorbinil. Ascorbic acid also prevented the reduction in myo-inositol uptake due to excess extracellular glucose, at 3 and 30 microM concentrations, but not at 300 microM. Neither sorbinil nor ascorbic acid could prevent the alterations in myo-inositol transport in cells exposed to high sorbitol levels for the same period of time. These data suggest that glucose-induced alteration of myo-inositol transport in Schwann cells is mediated, at least in part, via sorbitol accumulation. This myo-inositol transport impairment is prevented by sorbinil and also by ascorbic acid. Ascorbic acid may hold a fresh promise for the treatment/prevention of diabetic neuropathy/complications, at least as an adjunct therapy along with known aldose reductase inhibitors.

Modulation of basal nitric oxide-dependent cyclic-GMP production by ambient glucose, myo-inositol, and protein kinase C in SH-SY5Y human neuroblastoma cells.

Defective tissue perfusion and nitric oxide production and altered myo-inositol metabolism and protein kinase C activation have been invoked in the pathogenesis of diabetic complications including neuropathy. The precise cellular compartmentalization and mechanistic interrelationships of these abnormalities remain obscure, and nitric oxide possesses both neurotransmitter and vasodilator activity. Therefore the effects of ambient glucose and myo-inositol on nitric oxide-dependent cGMP production and protein kinase C activity were studied in SH-SY5Y human neuroblastoma cells, a cell culture model for peripheral cholinergic neurons. D-Glucose lowered cellular myo-inositol content, phosphatidylinositol synthesis, and phosphorylation of an endogenous protein kinase C substrate, and specifically reduced nitric oxide-dependent cGMP production a time- and dose-dependent manner with an apparent IC50 of approximately 30 mM. The near maximal decrease in cGMP induced by 50 mM D-glucose was corrected by the addition of protein kinase C agonists or 500 microM myo-inositol to the culture medium, and was reproduced by protein kinase C inhibition or downregulation, or by myo-inositol deficient medium. Sodium nitroprusside increased cGMP in a dose-dependent fashion, with low concentrations (1 microM) counteracting the effects of 50 mM D-glucose or protein kinase C inhibition. The demonstration that elevated D-glucose diminishes basal nitric oxide-dependent cGMP production by myo-inositol depletion and protein kinase C inhibition in peripheral cholinergic neurons provides a potential metabolic basis for impaired nitric oxide production, nerve blood flow, and nerve impulse conduction in diabetes.

Pregnancy rates relating to plasma progesterone levels in recipient heifers at day of transfer.

A group of Holstein heifers (n=223), weighing approximately 454 kg, were used to determine pregnancy rates in relation to plasma progesterone concentrations in recipients on the day of embryo transfer. All recipients were in estrus within +/- 12 hours of the donor cows. These data showed a cubic trend by regression analysis. Chi-square test revealed that there was a significant (P<0.0001) relationship between plasma progesterone concentrations and resulting pregnancies. Pregnancy rates were low when plasma progesterone concentrations were below 2.00 ng/ml. Actual number of pregnancies relating to specific plasma progesterone groups were 12 61 (20%) for <2.00 ng/ml, 94 127 (74%) for concentrations between 2.00 and 5.00 ng/ml, and 21 35 (60%) for >5.00 ng/ml. Corpora lutea were classified as good, poor, or cystic by both manual and visual observation. These observations revealed that manual palpation of the corpus luteum was not a valid criterion of the corpus luteum function as measured by plasma progesterone concentrations. Further observation revealed no significant relationship between plasma progesterone and whether the corpus luteum was on the left or right ovary. Hence, pregnancy rate was not significantly associated with the left or right ovary. Pregnancies were determined by rectal palpation at 60 days.