Identification and characterization of newtrad (ras associated with diabetes), a gene specifically expressed in regenerating limb muscle

Abstract

Formation of the blastema is a key event for limb regeneration in urodele amphibians, and skeletal muscle has been thought to be a major origin of the multipotent blastemal mesenchyme. In the present study, we used differential display to identify the genes expressed differentially in the muscle at the amputation site. We have isolated a cDNA clone that was upregulated during limb regeneration of the Japanese newt, Cynops pyrrhogaster. Deduced amino acid sequence revealed that the cloned cDNA was a newt homolog of rad (ras associated with diabetes), a gene overexpressed in skeletal muscle of Type II diabetic patients. Expression of newt rad (nrad) was not observed in unamputated normal limb muscle, increased within 4 hr after amputation, and then decreased to the level of normal muscle between 11 and 21 days after amputation. In situ hybridization showed that the transcripts of nrad were localized around most of the nuclei of skeletal muscle near the amputation site, indicating the expression of nrad in the multinucleate myotubes. This expression gradually decreased along the distal to proximal axis. No signals were observed in apical epidermal cap or blastemal mesenchyme. However, reverse transcription‐PCR analysis detected a very low level of nrad expression in blastema, suggesting the carry‐over of nrad expression in blastema from muscle. Administration of retinoic acid, which has been shown to cause an enhanced dedifferentiation in the regenerating limbs, increased nrad expression in more proximally located limb muscle tissues and prolonged the expression period. Thus, it was strongly suggested that the nrad expression is correlated with the dedifferentiation of myotubes of regenerating limbs. We also analyzed the expression of nrad during development. Transcripts were observed in immature oocytes, seen faintly or not seen thereafter until stage 57 when its expression increased again. These results indicated that nrad may play a role(s) in the developmental process as well as limb regeneration. © 2001 Wiley‐Liss, Inc.

Proteomic analysis of blastema formation in regenerating axolotl limbs

“…Dedifferentiated cells express a number of genes associated with the dedifferentiated state, such as msx1 [129], Nrad [130], rfrng and notch [131]. Nuclear transplantation studies [132] and ectopic grafting experiments [133] have shown that blastema cells are not reprogrammed to pluripotency.…”

Overexpression of Rad in muscle worsens diet-induced insulin resistance and glucose intolerance and lowers plasma triglyceride level

“…Rad expression in vascular smooth muscle cells is highly induced by platelet-derived growth factor, TNF-␣, and balloon injury of the endothelium (12). Rad is also up-regulated in regenerating limb muscle of the newt (19), in some human breast cancers (20), and in human peripheral blood mononuclear cells in response to acute heat shock (21). Thus, there are a number of normal and pathological situations in which Rad expression is increased.…”

The RGK family of GTP-binding proteins: Regulators of voltage-dependent calcium channels and cytoskeleton remodeling

“…Regulation may result from the hyperinsulinemia associated with the disease [40] as insulin has been shown to increase Rad mRNA levels in muscle [25]. In addition, Rad expression is upregulated in both developing and post-amputation de-differentiating muscle cells in the newt [33]. A similar regulation occurs in developing and regenerating muscle [20,24], as well as in denervated or injured muscle [20,27], and in vascular smooth muscle cells following balloon injury [21].…”

“…Among the most unique features of RGK proteins is their ability to be transcriptionally regulated [3,5,6,11,[20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] (Table 2). Indeed, Gem/Kir was discovered as a gene upregulated in human T cells after stimulation with mitogens [5] or in BCR-Abl-transformed B cells [11].…”

“…The other RGK subfamily members also demonstrate tissue and molecule-specific transcriptional regulation [3,4,6,20,21,24,25,27,[30][31][32][33][34][35]. Rem is expressed predominantly in cardiac muscle, but also at more modest levels in lung, kidney, and skeletal muscle [3]; Rem2 is highly expressed in the brain and kidney, but also in neuroendocrine tissues [4]; Rad is found in abundance in cardiac and skeletal muscle [6]; while Gem/Kir is found in a diverse set of tissues, including myeloid cells, kidney, liver, and lung [5].…”