大鼠神经轴索损伤erbB3 mRNA的表达
作者：李明 苏姗 史蒂文.谢勒
单位：李明(昆明医学院解剖教研室，昆明 650031)；苏姗(宾夕法尼亚大学医学院神经科，美国 19104-6146)；史蒂文.谢勒(宾夕法尼亚大学医学院神经科，美国 19104-6146)
关键词：erbB3；轴突与雪旺细胞的相互作用；轴突切断；坐骨神经
昆明医学院学学报000106The Expression of ErbB3/Neu mRNA in Rat Axotomized Nerves
LI Ming
(epartment of Anatomy Kunming Medical College，Kunming 650031;)
Suan SHUNMAS Steven S.SCHERER
, 百拇医药
(Department of Neurology Clinical Research Building，The University of Pennsylvania Philadelphia，PA 19104-6146 )
Abstract：Neuregulins are a family of peptide growth factors with potent effects on Schwann cells. We have investigated the regulation of erbB3/neu，which form the neuregulin receptor of rat Schwann cells. In the adult sciatic nerve，the expression of erbB3 mRNA increases in parallel following axotomy. ErbB3 mRNA is expressed by denervated Schwann cells in axotomized nerves.So，the expression of erbB3 mRNA is regulated by axon-Schwann cell interactions.
, 百拇医药
Key words：erbB3;axon-Schwann cell interactions;axotomy;rat nerve
CLC number：Q 189;Q 421 Document code：A Article ID：1003-4706(2000)01-0025-04
摘 要：神经生长调节因子(Neuregulins)是一个多肽生长因子的家族，它对神经雪旺氏细胞具有重要作用. 我们调查了大鼠雪旺细胞神经生长因子受体的调节作用，发现在成年鼠的坐骨神经中，erbB3 mRNA的表达随神经轴索损伤程度而增加，通过去除神经轴索上的雪旺细胞表达增加. 结论：神经生长调节因子erbB3 mRNA受体的表达是通过轴索-雪旺氏细胞的相互作用调节完成.
Neuregulins are a family of peptide growth factors expressed by neurons and mesenchymal cells[1，2]. Neuregulins were independentiy discovered by several laboratories，leading to a number of different names，glial growth factor (GGF)，neu differentiation factor (NDF)，acetylcholine receptor-inducting activity (ARIA)，and heregulin(HRG). The purification of GGF led to the cloning of the neuregulin gene and the realization that there were multiple isoforms，including cytoplasmic，transmembrane，and secreted isoforms，resulting from alternative splicing[3]. One of alternatively splices affects the EGF domain，creating the so-called α and β isoforms，which differ dramatically in their ability to promote the survival and proliferation of Schwann cell(SCs)[4].
, http://www.100md.com
Sensory and motor neurons express and axonally transport neuregulins from early embryonic stages[3，5]，so that SCs are probably continuous exposed to neuregulins throughout their development. Neuregulin recruit neural crest cells into the SC lineage[6]，and serve as survival and mitogens for SCs and their precursors[4，7，8，9]. As might be expected from the multiple effects of neuregulins，mice with a disrupted neuregulin gene are deficient in SCs[10].
, http://www.100md.com
The receptors for the neuregulins are members of the erbB family of receptor tyrosine kinases[1，2]. ErbB3 can bind neuregulins but has a defective kinase domain. SCs express erbB3，and it is phosphorylated in response to neuregulins[11，12].
To understand the expression of erbB3 mRNA following axonal lesion in the adult sciatic nerve. We have investigated the expression of erbB3 mRNA in rat axotomized nerves.
, http://www.100md.com 1 Materials and Methods
1.1 Surgery and collection of tissues
Using aseptic technique，the sciatic nerves of anesthetized (50 mg/kg，pentobarbital i.p.)，adult (10～13 week old) Sprague-Dawley rats were axotomized. Transecting and suturing the two nerve-stumps were at least I cm apart，this technique prevents axonal regeneration to the distal nerve-stump for at least 2 months. Nerve-crush was produced by tightly compressing the sciatic nerve at the sciatic notch with flattened forceps twice，each time for 10 seconds，this technique causes all of the axons to degenerate，but allows axonal regeneration. At various times after nerve-injury，the animals were sacrificed by CO2 inhalation，the distal nerve-stumps were removed. For transected nerves，the entire distal nerve-stump was taken from just below the lesion to the ankle (about 4 cm). For crushed nerves，the distal nerve-stump was divided into the proximal and distal segments，each about 2cm long，from P4 (P1 is the first day after birth) to P58. The nerves were immediately frozen in liquid nitrogen and stored at -80℃. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
, 百拇医药
1.2 Northern blotting
RNA was isolated from rat sciatic nerves and SCs by CsC12 gradient centrifugation. Equal amounts (10 μg) of total RNA were electrophoresed and transferred to nylon membranes，and u.v. cross-linked (0. 12 joules). Blots were prehybridized，hybridized，and washed using standard techniques. The following cDNAs were used as probes - a 2.3 kb fragment of rat erbB3，a 0.7 kb BamHI fragmann of rat NGFR，a full-length cDNA of rat Po and a full-length cDNA of rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Plasmid inserts were isolated after restriction endonuclease digestion and purified by electroelution. 32P-labeled cDNA probes with specific activities of 2～5 x 109 cpm/g were prepared.
, 百拇医药
1.3 RNAse protection
Single-stranded anti-sense transcripts of rat erbB3 and rat GAPDH were generated with the appropriate RNA polymerase. Equal amounts(10 μg)of total RNA from rat sciatic nerves and rat SCs were incubated with 100 000 cpm of the erbB3，and 25 000 cpm of the GAPDH riboprobes. The RNA was denatured at 85℃ for 10min，hybridized overnight at 48℃，then digested with RNase TI and RNase A (final concentrations 1 μg /μL and 40 μg /mL，respectively) for 1 hour at 30℃. The reaction was stopped by adding proteinase K (final concentrations 0.28 μg /μL) for 30 min. at 37℃. The RNA was purified by phenol-chloroform extraction and precipitation with LiCI，yeast tRNA，and 100% ethanol. The RNA pellet was resuspended in loading dye，counted，and the protected fragments were separated on a sequencing gel.
, http://www.100md.com
2 Results
Figure 1 shows a northern blot analysis of erbB3 mRNA expression in transection axotomy. There was a low level of erbB3 in unlesioned nerves，but the level increases sharply at 4 days post-transection and remains elevated for at least 58 days. In addition，we also examined the level of erbB3 mRNA following nerve-crush. As shown in Fig 1，the increases of erbB3 mRNA were from a proximal segment D1 at 8 days，and remains elevated until 58 days.
, 百拇医药
Figure 1 Northern blot analysis of erbB3 in normal and lesioned adult rat sciatic nerve.
We confirmed the time course of erbB3 mRNA expression in lesioned nerves by performing an RNase protection analysis. As shown in Fig 2. the level increase of erbB3 mRNA was from 4 days to 58 days in the both of transection and crush，and sharply from 8 days to 58 days in transection as northern blot. The increase of erbB3 mRNA levels was higher following nerve-transection than nerve-crush from 8 days.
, 百拇医药
Figure 2 RNase protection assay of erbB3 mRNA in normal and lesioned adult rat sciatic nerve.
Eash lane contains an equal amount (10 μg) of total RNA isolated from the distal stumps of sciatic nerves that had been transected or crushed. The number of days after each of these lesions in indicated; the '0'time point is from unlesioned nerves. In crushed nerves，the distal nerve-stumps were divided into proximal D1 and distal D2 segments of equal lengths. The blots were successively hybridized with a radiolabeled cDNA probe for erbB3，NGFR，GAPDH，and PO.
, 百拇医药
Each lane contains an equal amount (10 μg) of total RNA isolated from the distal stumps of sciatic nerves that had been transected or crushed (see Fig 2 for details). The RNA was hybridized with antisense RNA for erbB3 and GAPDH，and exposed to film for 3 days erbB3 or 12 hours (GAPDH).
3 Discussion
In this report，we have investigated the expression of erbB3 in rat axotomized adult nerve，their expression increase in parallel in the SCs distal to the site of injury，but declines as SCs remyelinate regenerating axons. The denervated SCs created by axotomy also express erbB3[11]. We examined the level of erbB3 mRNA following nerve-crush，which also causes Wallerian degeneration，but allows the rapid regeneration of axons. To facilitate the analysis of gene expression that is regulated by axon-Schwann cell interactions，the distal nerve-stump was divided into two segments，a more proximal one D1 and a more distal one D2. Since axons regenerate in a proximal to distal manner，these changes should be evident first in the D1 segment，and subsequently in the D2 segment. We attribute this to the regeneration of axons into the D1 segment by 8 days and the D2 segment by 12 days following nerve-chush. The increase in erbB3 mRNA at 4 days post-crush is not affected，as regenerating axons have not reached the distal nerve-stump，but the later increase in erbB3 mRNA is also less than that in transected nerves at comparable times. Reprobing the blot for NGFR and Po mRNA demonstrates that axon-SC interactions affect the pattem of gene expression in crushed nerves. The increase in NGFR mRNA in crushed nerves parallels the increase in transected nerves，but is blunted by axon-SC interactions. The level of P0 mRNA，in contrast，returns towards normal in crushed nerves as SCs remyelinate regenerating axons.
, http://www.100md.com
Acknowledgements We thank Dr.Steven Burden for the riboprobe constructs for erbB3 and Dr.John Koland for erbB3 cDNA. ■
基金项目：Foundation itm:This work was supported by grants from the NIH (NS08075，NS01565)
作者简介：李明(1949-)，男，浙江人，副教授，硕士生导师，主要从事神经分子生物学和体质人类学研究.
References：
[1] CARRAWAY K L I，BURDEN S J. Neuregulins and their receptors[J]. Curr Opin Neurobiology，1995，5 : 606
, 百拇医药
[2] LEMKe G. Neuregulins in development[J]. Mol Cell Neurosci ，1996，7:247
[3] MARCHIONNI M A，GOODEARL A D J，CHEN M S,et al.Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system[J]. Nature，1993，362: 312
[4] DONG Z，BRENNAN A，LIU N，et al. Neu differentiation factor acts as a neuron-glia signal and regulates the survival，proliferation and maturation of the rat Schwann cell precursor[J]. Neuron，1995，585
, http://www.100md.com
[5] CORFAS G，ROSEN K M，ARATAKE H，et al. Differential expression of ARIA isoforms in the rat brain[J]. Neuron，1995，14: 1 03
[6] SHAH N M，MARCHIONNI M A，LSSACS I，et al.Glial growth factor restricts mammalian neural crest stem cells to a glial fate[J]. Cell，1994，77: 349
[7] GRINSPAN J B，MAREHIONNI M，REEVES M，et al.Axonal interaction regiilate Schwann cell apoptosis in developing peripheral nerve neuregulin receptors and the role of neuregulins[J]. J Neuroscience，1996，16: 6107
, http://www.100md.com
[8] TRACHTENBERG I T，THOMPSON W J. Schwann cell apoptosis and its regulation at the developing neuromuscular junction[J]. Nature，1996，379: 174
[9] WEINMASTER G，LEMKE G. Cell-speciTic cyclic-AMP mediated induction of the PDGF receptor[J]. EMBO J，1990，9: 915
[10] MEYER D，BIRCHMEIER C. Multiple essential functions of neurogulin in development[J]. Nature，1995，378: 386
[11] COHEN J A，YACHNIS A T，ARAI M，et al. Expression of the neu proto-oncogene by Schwann cells during peripheral nerve development and Wallerian degeneration[J]. J Neuroscience Res，1992，31: 622
收稿日期：2000-01-05, http://www.100md.com
单位：李明(昆明医学院解剖教研室，昆明 650031)；苏姗(宾夕法尼亚大学医学院神经科，美国 19104-6146)；史蒂文.谢勒(宾夕法尼亚大学医学院神经科，美国 19104-6146)
关键词：erbB3；轴突与雪旺细胞的相互作用；轴突切断；坐骨神经
昆明医学院学学报000106The Expression of ErbB3/Neu mRNA in Rat Axotomized Nerves
LI Ming
(epartment of Anatomy Kunming Medical College，Kunming 650031;)
Suan SHUNMAS Steven S.SCHERER
, 百拇医药
(Department of Neurology Clinical Research Building，The University of Pennsylvania Philadelphia，PA 19104-6146 )
Abstract：Neuregulins are a family of peptide growth factors with potent effects on Schwann cells. We have investigated the regulation of erbB3/neu，which form the neuregulin receptor of rat Schwann cells. In the adult sciatic nerve，the expression of erbB3 mRNA increases in parallel following axotomy. ErbB3 mRNA is expressed by denervated Schwann cells in axotomized nerves.So，the expression of erbB3 mRNA is regulated by axon-Schwann cell interactions.
, 百拇医药
Key words：erbB3;axon-Schwann cell interactions;axotomy;rat nerve
CLC number：Q 189;Q 421 Document code：A Article ID：1003-4706(2000)01-0025-04
摘 要：神经生长调节因子(Neuregulins)是一个多肽生长因子的家族，它对神经雪旺氏细胞具有重要作用. 我们调查了大鼠雪旺细胞神经生长因子受体的调节作用，发现在成年鼠的坐骨神经中，erbB3 mRNA的表达随神经轴索损伤程度而增加，通过去除神经轴索上的雪旺细胞表达增加. 结论：神经生长调节因子erbB3 mRNA受体的表达是通过轴索-雪旺氏细胞的相互作用调节完成.
Neuregulins are a family of peptide growth factors expressed by neurons and mesenchymal cells[1，2]. Neuregulins were independentiy discovered by several laboratories，leading to a number of different names，glial growth factor (GGF)，neu differentiation factor (NDF)，acetylcholine receptor-inducting activity (ARIA)，and heregulin(HRG). The purification of GGF led to the cloning of the neuregulin gene and the realization that there were multiple isoforms，including cytoplasmic，transmembrane，and secreted isoforms，resulting from alternative splicing[3]. One of alternatively splices affects the EGF domain，creating the so-called α and β isoforms，which differ dramatically in their ability to promote the survival and proliferation of Schwann cell(SCs)[4].
, http://www.100md.com
Sensory and motor neurons express and axonally transport neuregulins from early embryonic stages[3，5]，so that SCs are probably continuous exposed to neuregulins throughout their development. Neuregulin recruit neural crest cells into the SC lineage[6]，and serve as survival and mitogens for SCs and their precursors[4，7，8，9]. As might be expected from the multiple effects of neuregulins，mice with a disrupted neuregulin gene are deficient in SCs[10].
, http://www.100md.com
The receptors for the neuregulins are members of the erbB family of receptor tyrosine kinases[1，2]. ErbB3 can bind neuregulins but has a defective kinase domain. SCs express erbB3，and it is phosphorylated in response to neuregulins[11，12].
To understand the expression of erbB3 mRNA following axonal lesion in the adult sciatic nerve. We have investigated the expression of erbB3 mRNA in rat axotomized nerves.
, http://www.100md.com 1 Materials and Methods
1.1 Surgery and collection of tissues
Using aseptic technique，the sciatic nerves of anesthetized (50 mg/kg，pentobarbital i.p.)，adult (10～13 week old) Sprague-Dawley rats were axotomized. Transecting and suturing the two nerve-stumps were at least I cm apart，this technique prevents axonal regeneration to the distal nerve-stump for at least 2 months. Nerve-crush was produced by tightly compressing the sciatic nerve at the sciatic notch with flattened forceps twice，each time for 10 seconds，this technique causes all of the axons to degenerate，but allows axonal regeneration. At various times after nerve-injury，the animals were sacrificed by CO2 inhalation，the distal nerve-stumps were removed. For transected nerves，the entire distal nerve-stump was taken from just below the lesion to the ankle (about 4 cm). For crushed nerves，the distal nerve-stump was divided into the proximal and distal segments，each about 2cm long，from P4 (P1 is the first day after birth) to P58. The nerves were immediately frozen in liquid nitrogen and stored at -80℃. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
, 百拇医药
1.2 Northern blotting
RNA was isolated from rat sciatic nerves and SCs by CsC12 gradient centrifugation. Equal amounts (10 μg) of total RNA were electrophoresed and transferred to nylon membranes，and u.v. cross-linked (0. 12 joules). Blots were prehybridized，hybridized，and washed using standard techniques. The following cDNAs were used as probes - a 2.3 kb fragment of rat erbB3，a 0.7 kb BamHI fragmann of rat NGFR，a full-length cDNA of rat Po and a full-length cDNA of rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Plasmid inserts were isolated after restriction endonuclease digestion and purified by electroelution. 32P-labeled cDNA probes with specific activities of 2～5 x 109 cpm/g were prepared.
, 百拇医药
1.3 RNAse protection
Single-stranded anti-sense transcripts of rat erbB3 and rat GAPDH were generated with the appropriate RNA polymerase. Equal amounts(10 μg)of total RNA from rat sciatic nerves and rat SCs were incubated with 100 000 cpm of the erbB3，and 25 000 cpm of the GAPDH riboprobes. The RNA was denatured at 85℃ for 10min，hybridized overnight at 48℃，then digested with RNase TI and RNase A (final concentrations 1 μg /μL and 40 μg /mL，respectively) for 1 hour at 30℃. The reaction was stopped by adding proteinase K (final concentrations 0.28 μg /μL) for 30 min. at 37℃. The RNA was purified by phenol-chloroform extraction and precipitation with LiCI，yeast tRNA，and 100% ethanol. The RNA pellet was resuspended in loading dye，counted，and the protected fragments were separated on a sequencing gel.
, http://www.100md.com
2 Results
Figure 1 shows a northern blot analysis of erbB3 mRNA expression in transection axotomy. There was a low level of erbB3 in unlesioned nerves，but the level increases sharply at 4 days post-transection and remains elevated for at least 58 days. In addition，we also examined the level of erbB3 mRNA following nerve-crush. As shown in Fig 1，the increases of erbB3 mRNA were from a proximal segment D1 at 8 days，and remains elevated until 58 days.
, 百拇医药
Figure 1 Northern blot analysis of erbB3 in normal and lesioned adult rat sciatic nerve.
We confirmed the time course of erbB3 mRNA expression in lesioned nerves by performing an RNase protection analysis. As shown in Fig 2. the level increase of erbB3 mRNA was from 4 days to 58 days in the both of transection and crush，and sharply from 8 days to 58 days in transection as northern blot. The increase of erbB3 mRNA levels was higher following nerve-transection than nerve-crush from 8 days.
, 百拇医药
Figure 2 RNase protection assay of erbB3 mRNA in normal and lesioned adult rat sciatic nerve.
Eash lane contains an equal amount (10 μg) of total RNA isolated from the distal stumps of sciatic nerves that had been transected or crushed. The number of days after each of these lesions in indicated; the '0'time point is from unlesioned nerves. In crushed nerves，the distal nerve-stumps were divided into proximal D1 and distal D2 segments of equal lengths. The blots were successively hybridized with a radiolabeled cDNA probe for erbB3，NGFR，GAPDH，and PO.
, 百拇医药
Each lane contains an equal amount (10 μg) of total RNA isolated from the distal stumps of sciatic nerves that had been transected or crushed (see Fig 2 for details). The RNA was hybridized with antisense RNA for erbB3 and GAPDH，and exposed to film for 3 days erbB3 or 12 hours (GAPDH).
3 Discussion
In this report，we have investigated the expression of erbB3 in rat axotomized adult nerve，their expression increase in parallel in the SCs distal to the site of injury，but declines as SCs remyelinate regenerating axons. The denervated SCs created by axotomy also express erbB3[11]. We examined the level of erbB3 mRNA following nerve-crush，which also causes Wallerian degeneration，but allows the rapid regeneration of axons. To facilitate the analysis of gene expression that is regulated by axon-Schwann cell interactions，the distal nerve-stump was divided into two segments，a more proximal one D1 and a more distal one D2. Since axons regenerate in a proximal to distal manner，these changes should be evident first in the D1 segment，and subsequently in the D2 segment. We attribute this to the regeneration of axons into the D1 segment by 8 days and the D2 segment by 12 days following nerve-chush. The increase in erbB3 mRNA at 4 days post-crush is not affected，as regenerating axons have not reached the distal nerve-stump，but the later increase in erbB3 mRNA is also less than that in transected nerves at comparable times. Reprobing the blot for NGFR and Po mRNA demonstrates that axon-SC interactions affect the pattem of gene expression in crushed nerves. The increase in NGFR mRNA in crushed nerves parallels the increase in transected nerves，but is blunted by axon-SC interactions. The level of P0 mRNA，in contrast，returns towards normal in crushed nerves as SCs remyelinate regenerating axons.
, http://www.100md.com
Acknowledgements We thank Dr.Steven Burden for the riboprobe constructs for erbB3 and Dr.John Koland for erbB3 cDNA. ■
基金项目：Foundation itm:This work was supported by grants from the NIH (NS08075，NS01565)
作者简介：李明(1949-)，男，浙江人，副教授，硕士生导师，主要从事神经分子生物学和体质人类学研究.
References：
[1] CARRAWAY K L I，BURDEN S J. Neuregulins and their receptors[J]. Curr Opin Neurobiology，1995，5 : 606
, 百拇医药
[2] LEMKe G. Neuregulins in development[J]. Mol Cell Neurosci ，1996，7:247
[3] MARCHIONNI M A，GOODEARL A D J，CHEN M S,et al.Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system[J]. Nature，1993，362: 312
[4] DONG Z，BRENNAN A，LIU N，et al. Neu differentiation factor acts as a neuron-glia signal and regulates the survival，proliferation and maturation of the rat Schwann cell precursor[J]. Neuron，1995，585
, http://www.100md.com
[5] CORFAS G，ROSEN K M，ARATAKE H，et al. Differential expression of ARIA isoforms in the rat brain[J]. Neuron，1995，14: 1 03
[6] SHAH N M，MARCHIONNI M A，LSSACS I，et al.Glial growth factor restricts mammalian neural crest stem cells to a glial fate[J]. Cell，1994，77: 349
[7] GRINSPAN J B，MAREHIONNI M，REEVES M，et al.Axonal interaction regiilate Schwann cell apoptosis in developing peripheral nerve neuregulin receptors and the role of neuregulins[J]. J Neuroscience，1996，16: 6107
, http://www.100md.com
[8] TRACHTENBERG I T，THOMPSON W J. Schwann cell apoptosis and its regulation at the developing neuromuscular junction[J]. Nature，1996，379: 174
[9] WEINMASTER G，LEMKE G. Cell-speciTic cyclic-AMP mediated induction of the PDGF receptor[J]. EMBO J，1990，9: 915
[10] MEYER D，BIRCHMEIER C. Multiple essential functions of neurogulin in development[J]. Nature，1995，378: 386
[11] COHEN J A，YACHNIS A T，ARAI M，et al. Expression of the neu proto-oncogene by Schwann cells during peripheral nerve development and Wallerian degeneration[J]. J Neuroscience Res，1992，31: 622
收稿日期：2000-01-05, http://www.100md.com