Tateki Kubo

Mesenchymal–epithelial interactions in the skin: increased expression of dickkopf1 by palmoplantar fibroblasts inhibits melanocyte growth and differentiation

We investigated whether or not the topographic regulation of melanocyte differentiation is determined by mesenchymal–epithelial interactions via fibroblast-derived factors. The melanocyte density in palmoplantar human skin (i.e., skin on the palms and the soles) is five times lower than that found in nonpalmoplantar sites. Palmoplantar fibroblasts significantly suppressed the growth and pigmentation of melanocytes compared with nonpalmoplantar fibroblasts. Using cDNA microarray analysis, fibroblasts derived from palmoplantar skin expressed high levels of dickkopf 1 (DKK1; an inhibitor of the canonical Wnt signaling pathway), whereas nonpalmoplantar fibroblasts expressed higher levels of DKK3. Transfection studies revealed that DKK1 decreased melanocyte function, probably through β-catenin–mediated regulation of microphthalmia-associated transcription factor activity, which in turn modulates the growth and differentiation of melanocytes. Thus, our results provide a basis to explain why skin on the palms and the soles is generally hypopigmented compared with other areas of the body, and might explain why melanocytes stop migrating in the palmoplantar area during human embryogenesis.

CARM1 Regulates Proliferation of PC12 Cells by Methylating HuD

ABSTRACT HuD is an RNA-binding protein that has been shown to induce neuronal differentiation by stabilizing labile mRNAs carrying AU-rich instability elements. Here, we show a novel mechanism of arginine methylation of HuD by coactivator-associated arginine methyltransferase 1 (CARM1) that affected mRNA turnover of p21cip1/waf1 mRNA in PC12 cells. CARM1 specifically methylated HuD in vitro and in vivo and colocalized with HuD in the cytoplasm. Inhibition of HuD methylation by CARM1 knockdown elongated the p21cip1/waf1 mRNA half-life and resulted in a slow growth rate and robust neuritogenesis in response to nerve growth factor (NGF). Methylation-resistant HuD bound more p21cip1/waf1 mRNA than did the wild type, and its overexpression upregulated p21cip1/waf1 protein expression. These results suggested that CARM1-methylated HuD maintains PC12 cells in the proliferative state by committing p21cip1/waf1 mRNA to its decay system. Since the methylated population of HuD was reduced in NGF-treated PC12 cells, downregulation of HuD methylation is a possible pathway through which NGF induces differentiation of PC12 cells.

Bone marrow cells differentiate into wound myofibroblasts and accelerate the healing of wounds with exposed bones when combined with an occlusive dressing

Background  The usefulness of bone marrow cells in accelerating wound healing has not been evaluated despite increasing evidence that bone marrow contains mesenchymal stem cells that have multipotentiality to differentiate into various types of cells after they enter the microenvironment of a specific tissue (niche).

Analysis of genes induced in peripheral nerve after axotomy using cDNA microarrays.

One of the most striking features of neurons in the mature peripheral nervous system is their ability to survive and to regenerate their axons following axonal injury. To perform a comprehensive survey of the molecular mechanisms that underlie peripheral nerve regeneration, we analyzed a cDNA library derived from the distal stumps of post‐injured sciatic nerve which was enriched in non‐myelinating Schwann cells using cDNA microarrays. The number of up‐ and down‐regulated genes in the transected sciatic nerve was 370 and 157, respectively, of the 9596 spotted genes. In the up‐regulated group, the number of known genes was 216 and the number of expressed sequence tag (EST) sequences was 154. In the down‐regulated group, the number of known genes was 103 and that of EST sequences was 54. We obtained several genes that were previously reported to be involved in regeneration of the injured neurons, such as cathepsin D, ninjurin 1, tenascin C, and co‐receptor for glial cell line‐derived neurotrophic factor family of trophic factors. In addition to unknown genes, there seemed to be a lot of annotated genes whose role in nerve regeneration remains unknown.

Activation of Rho in the injured axons following spinal cord injury

Axons of the adult central nervous system have very limited ability to regenerate after injury. This inability may be, at least partly, attributable to myelin‐derived proteins, such as myelin‐associated glycoprotein, Nogo and oligodendrocyte myelin glycoprotein. Recent evidence suggests that these proteins inhibit neurite outgrowth by activation of Rho through the neurotrophin receptor p75NTR/Nogo receptor complex. Despite rapidly growing knowledge on these signals at the molecular level, it remained to be determined whether Rho is activated after injury to the central nervous system. To assess this question, we establish a new method to visualize endogenous Rho activity in situ. After treatment of cerebellar granular neurons with the Nogo peptide in vitro, Rho is spatially activated and colocalizes with p75NTR. Following spinal cord injury in vivo, massive activation of Rho is observed in the injured neurites. Spatial regulation of Rho activity may be necessary for axonal regulation by the inhibitory cues.

bFGF regulates PI3-kinase-Rac1-JNK pathway and promotes fibroblast migration in wound healing.

Fibroblast proliferation and migration play important roles in wound healing. bFGF is known to promote both fibroblast proliferation and migration during the process of wound healing. However, the signal transduction of bFGF-induced fibroblast migration is still unclear, because bFGF can affect both proliferation and migration. Herein, we investigated the effect of bFGF on fibroblast migration regardless of its effect on fibroblast proliferation. We noticed involvement of the small GTPases of the Rho family, PI3-kinase, and JNK. bFGF activated RhoA, Rac1, PI3-kinase, and JNK in cultured fibroblasts. Inhibition of RhoA did not block bFGF-induced fibroblast migration, whereas inhibition of Rac1, PI3-kinase, or JNK blocked the fibroblast migration significantly. PI3-kinase-inhibited cells down-regulated the activities of Rac1 and JNK, and Rac1-inhibited cells down-regulated JNK activity, suggesting that PI3-kinase is upstream of Rac1 and that JNK is downstream of Rac1. Thus, we concluded that PI3-kinase, Rac1, and JNK were essential for bFGF-induced fibroblast migration, which is a novel pathway of bFGF-induced cell migration.

The neurotrophin receptor p75NTR in Schwann cells is implicated in remyelination and motor recovery after peripheral nerve injury

The function of the p75NTR neurotrophin receptor (p75NTR) in nervous system regeneration is still controversial. Part of that controversy may be due to the fact that p75NTR is expressed by both neuronal and glial cell types and may have very distinct and even contradictory roles in each population. In this study, to elucidate the in vivo function of p75NTR in Schwann cells during remyelination after peripheral nerve injury, we established a new animal model for p75NTR‐deficient Schwann cell transplantation in nude mice. We performed quantitative assessments of the functional, histological, and electrophysiological recovery after sciatic nerve injury, and compared them with those of the p75NTR(+/+) Schwann cell transplanted animals. At 7–10 weeks after injury, the motor recovery in the p75NTR(−/−) Schwann cell transplanted animals was significantly impaired compared with that in the p75NTR(+/+) Schwann cell transplanted animals. The lower number of the retrogradely labeled motoneurons and the hypomyelination in the p75NTR(−/−) Schwann cell transplanted animals were evident at 6 and 10 weeks after injury. At 10 weeks after injury, the radial growth in the axon caliber was also impaired in the p75NTR(−/−) Schwann cell transplanted animals. Measurement of the amount of myelin proteins and the nerve conduction velocity at 10 weeks after injury reflected these results. In summary, the p75NTR expression in Schwann cells is important for remyelination process, and the motor recovery after injury is impaired due to impaired axonal growth, remyelination, and radial growth in the axon calibers.

Rapid healing of intractable diabetic foot ulcers with exposed bones following a novel therapy of exposing bone marrow cells and then grafting epidermal sheets.

Background  Diabetic foot ulcers with exposed bones commonly result in amputation.

P311 accelerates nerve regeneration of the axotomized facial nerve

In axotomized adult neurons, a process of axonal regrowth and re‐establishment of the neuronal function has to be activated. Developmentally regulated factors may be reactivated during neuronal regeneration. Here we identify a gene, previously designated P311, that is up‐regulated in the axotomized facial motoneurons. Ectopically expressed P311 localizes in the cytoplasm and the nucleus. Over‐expression of P311 induces p21WAF1/Cip1 expression, leading PC12 cells to differentiate and to have neuron‐like morphologies. Adenovirus‐mediated P311 gene transfer promotes neurite outgrowth of postnatal dorsal root ganglion neurons and embryonic hippocampal neurons in vitro. This effect is abolished by the activation of Rho kinase. P311 also facilitates nerve regeneration following facial nerve injury in vivo. Our data provide evidence that genes involved in the differentiation process contribute to the regeneration of injured mature neurons, and may provide a practical molecular target.

Axonal supercharging technique using reverse end-to-side neurorrhaphy in peripheral nerve repair: an experimental study in the rat model

OBJECT In an attempt to improve peripheral nerve repair, the influence of the addition of reverse end-to-side neurorrhaphy for an injured peripheral nerve was investigated in the rat sciatic nerve transection model. METHODS Twelve Sprague-Dawley rats were divided into two groups (six rats in each group). In Group I, the right sciatic nerve was cut at a point distal to the gluteal notch and repaired using end-to-end neurorrhaphy with four 10-0 nylon epineurial sutures. In Group II, after performing the same procedure as in Group I, the left sciatic nerve was cut distally and passed through a subcutaneous tunnel to the right side. The proximal stump of the left sciatic nerve was coapted to the epineurial window of the right sciatic nerve distal to the injured point in an end-to-side fashion using 10-0 nylon epineurial sutures. The effects were evaluated using analgesimeter recordings for the hind paw, electrophysiological tests, measurement of the muscle contraction force, a double-labeling technique, weight measurement and histological examination of the gastrocnemius muscle, histological examination of the bilateral sciatic nerves, and immunofluorescent staining. RESULTS Results from the many tests used to evaluate the reverse end-to-side neurorrhaphy technique indicated that functional recovery of the denervated target organs was promoted by axonal augmentation. CONCLUSIONS The reverse end-to-side neurorrhaphy technique could be useful in peripheral nerve repair.