Wataru Morikawa

The Activity of Soluble VCAM-1 in Angiogenesis Stimulated by IL-4 and IL-13

IL-13 is a multifunctional lymphokine sharing a number of biological properties with IL-4. We previously observed that IL-4 shows angiogenic activities in vitro as well as in vivo. In this study we examined the effect of IL-13 on angiogenesis in vitro and in vivo and also the underlying mechanisms. Human IL-13 significantly stimulated the formation of tube-like structures in collagen gels by human microvascular endothelial cells and bovine aortic endothelial cells by about 3-fold over the controls in the absence of the cytokines. Administration of murine IL-13 led to neovascularization when implanted in the rat cornea. Coadministration of neutralizing mAb to the IL-4R inhibited both tubular morphogenesis in vitro and activation of STAT6 induced by IL-4 or IL-13. Both IL-4 and IL-13 markedly increased mRNA levels of VCAM-1 in vascular endothelial cells, and the production of the soluble form of VCAM-1 was also stimulated in response to IL-4 or IL-13. Administration of anti-VCAM-1 Ab in vitro blocked tubular morphogenesis induced by IL-4 and IL-13. Angiogenesis induced in vivo in rat cornea by IL-4 and IL-13 was also inhibited by Ab against the rat α4 integrin subunit. These findings suggest that angiogenesis dependent on IL-4 and IL-13 is mainly mediated through a soluble VCAM-1/α4 integrin pathway.

Downregulation of Cap43 gene by von Hippel‐Lindau tumor suppressor protein in human renal cancer cells

We previously identified 9 genes (i.e., thymosin β4, secreted protein acidic and rich in cysteine, Cap43, ceruloplasmin, serum amyloid A, heat shock protein 90, LOT1, osteopontin and casein kinase Iγ) that are more highly expressed in cancerous regions than in noncancerous regions in human renal cancers. In our study, we considered the possibility that the von Hippel‐Lindau (VHL) tumor suppressor gene might be able to affect the expression of these 9 genes in renal cancer cells. We first established 2 VHL‐positive cell lines, 786/VHL‐1 and 786/VHL‐2, after the introduction of wild‐type VHL into VHL‐negative renal cancer 786‐O cells. Of these 9 genes, expression of the Cap43 gene was specifically downregulated by VHL. Expression of Cap43 was also much lower in 4 other VHL‐positive renal cancer cell lines than in VHL‐negative 786‐O cells. Cap43 promoter assays with several deletion or mutation constructs demonstrated that the Sp1 site in the element from –286 base pairs (bp) to –62 bp was partly responsible for VHL‐induced suppression of the Cap43 gene. Immunostaining analysis with human specimens of renal cancers demonstrated that the Cap43 protein was expressed in most cancer cells and macrophages. We also observed a marked and specific increase of Cap43 mRNA levels in response to hypoxia or nickel in all VHL‐positive cell lines. Cellular expression of Cap43 mRNA in response to hypoxia or nickel thus is closely associated with VHL gene expression in renal cancer cells. Although the function of the Cap43 protein remains unclear, the expression of Cap43 protein could be a molecular marker closely associated with VHL in renal cancer.

Cellular Distribution of NDRG1 Protein in the Rat Kidney and Brain During Normal Postnatal Development

N-myc downregulated gene 1 (NDRG1) is a 43-kD protein whose mRNA is induced by DNA damage, hypoxia, or prolonged elevation of intracellular calcium. Although NDRG1 is also upregulated during cell differentiation, there are few studies on NDRG1 expression during postnatal development. Here we investigated the expression and cellular distribution of NDRG1 protein in rat kidney and brain during postnatal development. Immunohistochemical analysis revealed that the cellular localization of NDRG1 protein in the kidney changed from the proximal convoluted tubules to the collecting ducts between postnatal days 10 and 20. In the brain, a change in cellular expression was also found from the hippocampal pyramidal neurons to the astrocytes in the gray matter during the same postnatal period. These alterations in the cellular distribution of NDRG1 were associated with shifts in the molecular assembly on Western blots. Under non-reduced conditions, the main NDRG1 band was found only around 215 kD in both kidney and brain during the early postnatal stage. After postnatal day 10, the immunoreactive bands shifted to 43 kD in the kidney and 129 kD in the brain. These changes in the cellular distribution and state of assembly may correlate with the functional maturation of both organs.

N-myc downstream-regulated gene 1 expression in injured sciatic nerves

N‐myc downstream‐regulated gene 1 (NDRG1)/RTP/Drg1/Cap43/rit42/TDD5/Ndr1 is expressed ubiquitously and has been proposed to play a role in growth arrest and cell differentiation. A recent study showed that mutation of this gene is responsible for hereditary motor and sensory neuropathy‐Lom. However, the role of this gene in the peripheral nervous system is not fully understood. In our study, rabbit polyclonal antibodies were raised against this gene product and were used to examine changes in its expression over the time course of Wallerian degeneration and ensuing regeneration after crush injury of mouse sciatic nerves. Fluorescent immunohistochemistry showed that NDRG1 was expressed over the intact nerve fibers. Double labeling with a Schwann cell (SC) marker, S‐100 protein (S‐100), revealed that NDRG1 was localized in the cytoplasm of S‐100‐positive Schwann cells (SCs). NDRG1 expression was maintained in the early stage of myelin degradation but was then markedly depleted at the end stage of myelin degradation when frequent occurrence of BrdU‐labeled SCs was observed (at 7–9 days). The depletion of NDRG1 at this time point was also confirmed by Western blotting analysis. NDRG1 expression finally recovered at the stage of remyelination, with immunoreactivity stronger than that in intact nerves. These findings suggest that NDRG1 may play an important role in the terminal differentiation of SCs during nerve regeneration.