Expression of Reg/PAP family members during motor nerve regeneration in rat

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Abstract

In this study, we examined the expression of mRNAs for Regenerating gene (Reg)/pancreatitis-associated protein (PAP) family members following hypoglossal nerve injury in rats. In addition to four rat family members (RegI, Reg-2/PAP I, PAP II, and PAP III) that had been identified, we newly cloned and sequenced a type-IV Reg gene in rats. Among these five family members, the expression of Reg-2/PAP I mRNA was predominantly enhanced in injured motor neurons after axotomy. Furthermore, a marked induction of PAP III mRNA was observed in the distal part of the injured nerve. A polyclonal antibody was raised against PAP III, and a Western blotting analysis using this antibody confirmed an increased level of PAP III protein in the injured nerve. These results suggest that Reg family members would be new mediators among injured neurons and glial cells, and may play pivotal roles during nerve regeneration.

Section snippets

Materials and methods

Animals and surgery. Adult male Wistar rats weighing 150–200 g (6–8 weeks of age) were used throughout this study after anesthesia with pentobarbital. They were placed in a supine position and their right hypoglossal nerves were then cut with a pair of scissors just proximal to the bifurcation at the hyoid bone. Experiments were carried out in accordance with the Guideline laid down by Osaka City University Medical School regarding the care and use of animals for experimental procedures.

In situ

mRNA expressions of Reg family in injured motor neurons

To date, four isoforms of Reg family member genes, RegI, PAP I, PAP II, and PAP III, have been cloned in rats (Table 1). To examine the expression of the complete set of Reg family mRNAs in rats, we attempted to clone the unidentified rat RegIV gene. Eventually we obtained and sequenced the rat ortholog of human or mouse RegIV/RELP (regenerating protein-like protein) [17], [18] and designated it as rat RegIV (GenBank Accession No.: AB164049 in Table 1). First, we examined the mRNA expressions

Discussion

In this study, we examined the alterations in rat Reg family member expressions after peripheral motor nerve injury, and revealed the cell type-specific induction of Reg family members in response to nerve injury. PAP I expression is predominantly induced in injured motor neurons, whereas PAP III expression is preferentially enhanced in injured nerve. These specific expressions of PAP I and PAP III in injured motor neurons and nerves, respectively, suggest that these Reg family proteins are

Acknowledgments

We thank Dr. Graf (University Hospital, Zürich) for donating pPIC9-PAP III and Dr. Henderson (Developmental Biology Institute, Marseille) for donating the hybridoma producing the anti-p75 antibody. We are also grateful to C. Kadono, I. Jikihara, and T. Iwasaki for technical assistance, and E. Fukui and T. Kawai for secretarial assistance. This study was supported in part by grants from the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Science, Technology, Sports and

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    First two authors are equally contributed to this report.

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