Reg1 gene is expressed in regenerating muscle

INTRODUCTION In 1988, Terazono et al. first isolated a gene expressed in regenerating pancreatic β cells which he named regenerating gene (Reg) (Terazono et al. 1988). Several reg and reg-related genes have been isolated from mammalian species and they constitute a multigene family, the Reg gene family. Based on the primary structure of the proteins encoded by Reg family genes, the members of the Reg family have been grouped into four subclasses, type I, II, III and IV (Okamoto 1999, Hartupee 2001). Reg1 was originally isolated from a rat regenerating islet-derived cDNA library (Terazono et al. 1988) and it acts as a growth factor for pancreatic β cells in an autocrine/paracrine manner (Okamoto 1999). Reg1 protein expression has also been found in the rat gastrointestinal tissues and kidney (Watanabe et al. 1993). Since there are no studies showing Reg1 expression in healthy or injured skeletal rat muscles, the aim of this investigation was to reveal the Reg1 gene expression in regenerating skeletal muscle. Previous studies reported that there was no expression of Reg1 gene after peripheral nerve axotomy. Therefore, in this study, we examined the expression of Reg1 gene of corresponding nerve in response to skeletal muscle injury. MATERIAL AND METHODS In two-month old Wistar rats regeneration process was induced in slow (musculus soleus, SOL) and fast (musculus extensor digitorum longus, EDL) type of muscle. Muscle regeneration was induced by injection of local anesthetic (0.5% bupivacain). Immunohistochemistry and PCR analysis where performed for detection of Reg 1 mRNA in untreated and regenerating SOL, EDL and in sciatic nerve after 6, 12, 24 hours, 3 and 7 days after regeneration process was induced. Immunohistochemical staining for anti-rat Reg 1 protein Mab. Tissue samples of rat muscles and nerves were fixed in cold acetone. The cryostat sections were incubated with an anti-rat Reg 1 protein antibody overnight at 4°C and with secondary antibody at room temperature for 30 min. Bound antibody was detected by universal immunoenzyme polymer method followed by hematoxylin staining. RT-PCR. Total RNA was extracted from samples using Promega Total RNA extraction kit (SV Total RNA Isolation system, Promega, Madison, WI, USA). 1000ng of total RNA from each sample was transcribed in cDNA using Superscript II RT (Invitrogen, Carlsbad, CA, USA). Reg1 primer sequences were 5'-TACAGCTGCCAATGTCTGGATT-3' and 5’-CAGTGTCCCAGGATTTGTAGAGA-3’. The amplification products were then separated by 3% agarose gel electrophoresis stained with Gel Red (Biotium, Hayward, CA, USA) and visualized under UV light. Results were quantified with densitometry method by «1D Image Analysis Software» (Eastman Kodak Company, Rochester, NY, USA). RESULTS Immunohistochemical analysis of SOL and EDL muscles after three days of regeneration process was induced showed Reg I positive cells. (Figure 1, 2) RT-PCR analysis showed that Reg1 gene is expressed in both types of skeletal muscles and sciatic nerve. EDL has a peek expression 12 hours after regeneration process was induced and the signal intensity was stronger but not statistically significant compeering to SOL. After 12 hours the signal intensity was rapidly decreased till the 3rd day. This time point has importance because of statistically significant difference in signal between SOL and EDL. Signal is weak but constant till 7th day. SOL has also peek expression 12 hours after injury, although the signal is more constant and is weakening until the day 7th. Sciatic nerve for instance has no detectable expression 6 hours after injury and first measurable signal has been detected after 12 hours with peek expression 24 hours after the local anesthetic has been applied. After 24 hours the signal is weakening and could not be detected 7 days after regeneration process was induced.