Cobalamin deficiency-induced changes of epidermal growth factor (EGF)-receptor expression and EGF levels in rat spinal cord

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Research Report

Cobalamin deficiency-induced changes of epidermal growth factor (EGF)-receptor expression and EGF levels in rat spinal cord Elena Muttia,1 , Valerio Magnaghib,1 , Daniela Vebera , Alessandro Faronib , Salvatore Pecec , Pier Paolo Di Fiorec , Giuseppe Scalabrinoa,⁎ a

“Città Studi” Department, Laboratory of Neuropathology, University of Milan, Via Mangiagalli 31, I-20133 Milano, Italy Department of Endocrinology, University of Milan, Via Balzaretti 9, I-20133 Milano, Italy c Department of Medicine, Surgery, and Dentistry, University of Milan, FIRC Institute for Molecular Oncology Foundation, at the IFOM-IEO Campus, Via di Rudinì 8, I-20142 Milano, Italy b

A R T I C LE I N FO

AB S T R A C T

Article history:

We investigated the effect of cobalamin (Cbl) deficiency on epidermal growth factor

Accepted 16 December 2010

receptor (EGFR) mRNA levels in the spinal cord (SC) and liver of rats made Cbl-deficient

Available online 23 December 2010

(Cbl-D) by means of total gastrectomy or a Cbl-D diet, and simultaneously measured the levels of the epidermal growth factor (EGF). Both methods of inducing Cbl deficiency

Keywords:

decreased EGFR expression in the SC and liver. Cbl replacement treatment normalized or

Epidermal growth factor receptor

nearly so most of the abnormalities in EGFR expression in the totally gastrectomized (TGX)

Real-time PCR

rats at different times. The EGFR-immunostaining intensity decreased in the SC white

RNAse protection assay

matter of the Cbl-D rats and significantly increased in that of the TGX, Cbl-treated rats. EGF

Spinal cord

levels significantly increased in liver of TGX rats and in SC of 4-month TGX rats, and the

Vitamin B12

increases returned to almost normal levels after a postoperative 2-month administration of Cbl to TGX rats. These findings demonstrate that Cbl deficiency dysregulates the EGFREGF dyad in these tissues. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

We have previously demonstrated that epidermal growth factor (EGF) is one of the mediators of the myelinotrophic action of vitamin B12 (cobalamin, Cbl) in the rat central

nervous system (CNS) (Scalabrino et al., 2008). EGF synthesis is arrested when Cbl is chronically withdrawn by means of total gastrectomy (TG) or a Cbl-deficient (Cbl-D) diet, and the prolonged intracerebroventricular administration of EGF to totally gastrectomized (TGX) rats is as effective as Cbl in

⁎ Corresponding author. Fax: +39 02 50315338. E-mail address: [email protected] (G. Scalabrino). Abbreviations: EGF, epidermal growth factor; Cbl, cobalamin; CNS, central nervous system; TG, total gastrectomy; Cbl-D, Cbl-deficient; TGX, totally gastrectomized; SC, spinal cord; EGFR, epidermal growth factor receptor; RPA, RNase protection assay; rt-PCR, real-time PCR; ROI, regions of interest; UO, unoperated; LPT, laparotomized; NGF, nerve growth factor; LP, laparotomy; Taq, Thermus aquaticus; MGB, minor groove binder; Ct, cycle threshold; w/v, weight/volume; OD, optical densitometry 1 These authors contributed equally to this work. 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.12.056

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preventing the appearance of myelinolytic lesions (the neuropathological hallmarks of Cbl-D central neuropathy) in the spinal cord (SC) (Scalabrino et al., 1999, 2000). Furthermore, prolonged postoperative Cbl treatment restores EGF synthesis in the SC and other CNS areas of TGX rats (Scalabrino et al., 1999) and myelin lesions similar to those due to Cbl deficiency have been observed in the SC white matter of rats repeatedly treated intracerebroventricularly with anti-EGF antibodies (Scalabrino et al., 2000). However, the duration of the postoperative Cbl treatment that restores SC EGF synthesis must be longer than that of the postoperative EGF treatment that prevents the appearance of the myelinolytic lesions in the SC white matter because these lesions appear before the arrest of SC EGF synthesis in both types of Cbl-D rats (Scalabrino et al., 1999; Tredici et al., 1998). The members of the EGF family bind to and activate the EGF receptor (EGFR, also called ErbB1), a 170 kDa membranespanning protein (Liu and Neufeld, 2007; Sibilia et al., 2007; Wieduwilt and Moasser, 2008; Wong and Guillaud, 2004). EGFR transcripts have been found in the neurons, astrocytes, oligodendrocytes, and microglia of developing and adult mammalian CNS (Aguirre et al., 2007; Kornblum et al., 1998; Liu et al., 2006; Xian and Zhou, 2004), and several lines of evidence suggest that it is an important regulator of this development (Birecree et al., 1991; Sibilia et al., 1998; Wagner et al., 2006). EGFR overexpression also increases myelin thickness and the number of remyelinated axons in the CNS of a transgenic mouse strain after focal demyelination (Liu et al., 2006), and EGFR signalling is involved in the proliferation and/or differentiation of astrocytes as astrocytes devoid of EGFR have an impaired proliferation capacity (Sibilia et al., 1998) and undergo increased apoptosis (Wagner et al., 2006). Furthermore, EGFR is important in triggering the activation of quiescent astrocytes after various CNS injuries (Liu and Neufeld, 2007; Liu et al., 2006), and it is known that astrocyte activation is a key feature of both human and rat Cbl-D central neuropathy (Scalabrino, 2009). On the basis of the above, we used an RNase protection assay (RPA) and real-time PCR (rt-PCR) to investigate whether Cbl deficiency modifies EGFR mRNA expression in the SC and liver of both types of Cbl-D rats, and whether this expression normalized when the Cbl deficiency of the TGX rats was corrected by means of chronically administered Cbl injections. We also investigated whether Cbl deficiency modifies the intensity of EGFR immunohistochemistry in the SC white matter of Cbl-D rats, and whether the immunostaining intensity normalized after the correction of the Cbl-D status. Finally, we determined EGF levels in the SC and liver in order to verify whether any changes were related to the changes in EGFR mRNA levels. SC and liver were chosen because: (a) all express EGFR (Natarajan et al., 2007; Werner et al., 1988) and synthesize EGF (Marti et al., 1989; Mullhaupt et al., 1994; Scalabrino et al., 1999; Werner et al., 1988); (b) the SC is proportionally the most developed area in the flat map of the adult rat CNS (Swanson, 1995) and the part of the CNS in which myelin is most severely damaged by Cbl deficiency in humans and rats (Scalabrino, 2009); and (c) the liver is morphologically little or not affected by Cbl deficiency, but it accumulates the highest amounts of Cbl in the body.

2.

Results

2.1.

Effect of Cbl deficiency on SC levels of EGFR mRNA

RPA showed that these were markedly lower in the 2- and 4-month TGX rats in comparison with the appropriate controls (Fig. 1, top), with the difference from controls being greater after 4 months. In the rats fed a Cbl-D diet, the level was much lower only after 12 months. Cbl replacement treatment increased EGFR mRNA levels only in the 4-month TGX rats but without reaching control levels (Fig. 1, top). When evaluated by means of rt-PCR assay (Fig. 1, bottom), the levels were lower in the 2-month TGX rats and, to a greater extent, in the 4-month TGX rats. The chronic Cbl-D diet markedly reduced the level only after 8 months. The post-TG Cbl treatments were both ineffective in restoring normal levels (Fig. 1, bottom).

2.2.

Effect of Cbl deficiency on hepatic levels of EGFR mRNA

RPA showed that the decrease in EGFR mRNA was much greater in the 2-month TGX than in the 4-month TGX rats; a decrease in the rats fed with the Cbl-D diet was observed only after 8 months (Fig. 2, top). Cbl replacement treatment increased the EGFR mRNA levels only in the 2-month TGX rats (Fig. 2, top). When evaluated by means of rt-PCR assay, the levels were markedly decreased both 2 and 4 months after TG. The chronic Cbl-D diet markedly reduced the level only after

Fig. 1 – Histograms showing the effects of Cbl deficiency and Cbl replacement treatment on the levels of EGFR mRNA in rat SC. Each column represents the mean value of the number of animals given in parentheses. The control groups were LPT rats for the TGX rats, and UO rats for the rats on the Cbl-D diet. mo, month(s).

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hepatic levels (Table 1). Finally, there was an age-related decrease in EGF levels in the SC.

3.

Fig. 2 – Histograms showing the effects of Cbl deficiency and Cbl replacement treatments on the levels of EGFR mRNA in rat liver. Each column represents the mean value of the number of animals given in parentheses. The control groups were same as in Fig. 1. mo, month(s).

Discussion

The main findings of this study are that Cbl deficiency causes decreased changes (albeit quantitatively different) in EGFR expression in SC and liver of the rat. The fact that the changes in EGFR expression were also observed in the rats fed a Cbl-D diet exclude the possibility that the changes in the TGX rats may have been due to hormonal derangement and/or malabsorption. However, we observed only a partial recovery of EGFR expression in Cbl-D SC after postoperative Cbl treatment: this is not surprising, because a substantial percentage of patients with clinically confirmed severe Cbl deficiency do not respond or hardly respond to Cbl therapy (Savage and Lindenbaum, 1995). It is well known that EGFR expression is also regulated by various hormones (Moghal and Sternberg, 1999; Petkovich et al., 1987), vitamin D3 (Petkovich et al., 1987), and retinoids (Sah et al., 2002; Zheng et al., 1992). Our findings show that Cbl also regulates EGFR expression in the CNS and elsewhere, even though we do not know the molecular mechanism(s)

8 months (Fig. 2, bottom). Neither of the Cbl replacement treatments modified the changes in TGX rats (Fig. 2, bottom). Fig. 3 shows the typical RPA bands of SC and liver recorded from individual rats in each group.

2.3. Effect of Cbl deficiency on the intensity of EGFR immunohistochemistry staining in SC white matter Fig. 4 shows that Cbl deficiency (however induced) significantly decreased the intensity of EGFR staining in the regions of interest (ROI) of the SC white matter in comparison with controls (unoperated (UO) and laparotomized (LPT)), and postoperative Cbl replacement significantly increased the value in comparison with that of 2-month TGX, untreated rats. Fig. 5 shows typical histological photomicrographs of the EGFR-immunoreactive SC white matter of the differently treated rats. Parenthetically, staining intensity was also decreased in the morphologically unaffected white matter of the SC dorsal corticospinal tract of Cbl-D rats.

2.4.

Effect of Cbl deficiency on EGF levels in SC and liver

There was a marked increase in EGF levels in the SCs of the 4-month TGX rats and the rats fed a Cbl-D diet for 8 months, and in the livers of 2-month TGX rats (Table 1). There was a significant decrease in the livers of rats on a Cbl-D diet for 8 months (Table 1). Both Cbl treatments induced a marked decrease in SC levels in comparison with those observed in 2-month LPT rats and 4-month TGX rats (Table 1). Cbl replacement treatment for 2 months after TG tended to normalize

Fig. 3 – Expression of EGFR mRNAs as detected by RPA in SC and liver of control and Cbl-D rats. SC from a typical 2-month LPT (lane 3), a typical 2-month TGX (lane 4), and a typical 2-month TGX, Cbl-treated rat (lane 5); liver from a typical 2-month LPT (lane 6), a typical 2-month TGX (lane 7), and a typical 2-month TGX, Cbl-treated rat (lane 8). The undigested probes (640 bp of EGFR and 135 bp of 18S) are shown in lane 1. Yeast tRNA (5 μg; Sigma, St Louis, MO) was used as the negative control (lane 2).

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Table 1 – Effect of Cbl deficiency on EGF levels of rat SC and liver. Type of rats

EGF levels (pg/mg protein) SC

2-month LPT 2-month TGX 2-month TGX, Cbl-treated 4-month TGX 4-month TGX, Cbl-treated Controls (aged 8 months) On Cbl-D diet (8 months)

Fig. 4 – Histograms showing the effects of Cbl deficiency and postoperative Cbl replacement treatment on the intensity of EGFR immunohistochemistry staining in the posterior funiculi of SC white matter of the rat. Each column represents the mean intensity (shown as OD) of all of the ROI in the different experimental groups, and each vertical bar the SD. For details and statistical analysis, see the Section 4.8. mo, month(s).

responsible for this regulation. Furthermore, given that nerve growth factor (NGF) down-regulates EGFR levels in cultured cells (Liu et al., 2000), we cannot rule out the possibility that

0.42 ± 0.05 0.38 ± 0.06 0.23 ± 0.03 1.71 ± 0.10 0.20 ± 0.05 0.04 ± 0.01 0.07 ± 0.01

Liver (9) (9) (9) (9) (9) (9) (9)

0.06 ± 0.01 (6) 0.15 ± 0.04 a (6) 0.06 ± 0.01 (6) 0.10 ± 0.03 (6) 0.05 ± 0.03 b (6) 0.09 ± 0.01 (6) 0.05 ± 0.02 c (6)

Mean values ± SD. The number of animals in each experimental group is shown in parentheses. Details concerning post-TG Cbl treatments (schemes a and b) and EGF assay are given in Section 4. The statistical significance of the differences between each pair of means was evaluated by Scheffé's test. There is no significant difference in the values of the different organs between 2-month LPT, 4-month LPT rats, and unoperated rats of the same age (not shown). No statistical analysis of the data relating to the SC samples is possible because the nine samples for each type of rat came from a three pools of three samples each (see Section 4.9). a p < 0.01 or less vs. 2-month LPT rats. b p < 0.05 or less vs. 4-month TGX rats. c p < 0.05 or less vs. controls (8 months).

the increased NGF levels previously observed by us in the SCs and livers of 4-month TGX rats (Scalabrino et al., 2006; Veber et al., 2008) may play a role in down-regulating EGFR expression in SC at this time.

Fig. 5 – Photomicrographs showing the effect of Cbl deficiency on EGFR immunohistochemistry in rat SC white matter. Detailed view of a typical coronal cross-section of the white matter of the thoracic SC segment (posterior funiculi) of (A) a typical 2-month LPT, (B) a typical 2-month TGX, (C) a typical 2-month TGX, Cbl-treated rat, and (D) a typical rat fed a Cbl-D diet for 8 months. Note the decreased immunostaining in the white matter of the typical TGX rat and its increase after Cbl replacement. There are no significant differences in EGFR staining intensity between the different areas of the white matter in the SCs of UO and 2-month LPT rats, or between the different SC segments of the rats in the different experimental groups (not shown). Original magnification, ×10.

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The two methods used to evaluate EGFR expression occasionally gave temporally different results (e.g. in the SC of rats fed a Cbl-D diet for 8 months), likely due to the intrinsic characteristic of the methods utilized (i.e. real dynamic versus end-point analysis of transcription). Furthermore, other authors obtained different results in studies of gene expression using the same methods (Dufour et al., 1999, Schürmann et al., 2009), in which different primers and probe have been used in RPA and rt-PCR assay for evaluating same gene expression. Furthermore, the regulation of the EGFR-EGF system (Wong and Guillaud, 2004) seems to be partially abrogated during chronic Cbl deficiency, which decreased EGFR expression in the SC of 4-month TGX rats and the liver of 2-month TGX rats, both of which showed significantly increased EGF levels. These results disagree with those of other authors who found that EGFR synthesis was stimulated by EGF in cultured cells (Bjorge and Kudlow, 1987; Kudlow et al., 1986). However, although it is hazardous to extrapolate in vivo effects from in vitro data, it may be relevant that decreased EGFR expression parallels decreased EGF levels in the liver of rats fed a Cbl-D diet for 8 months, and so it is reasonable to conclude that Cbl deficiency-induced changes in EGFR expression are sometimes EGF-dependent and sometimes not. We observed also decreased EGFR-immunoreactive cell levels and unchanged EGF levels in SC of 2-month TGX rats: a substantially identical result has been previously reported by ourselves in SCs of 2-month TGX rats, which showed decreased p75immunoreactive cell levels and normal NGF levels (Scalabrino et al., 2006; Mutti et al., 2007). Furthermore, the increases in EGF levels in SC and liver of TGX rats are substantially reduced by postoperative Cbl replacement therapy, which leads us to conclude that they are related to Cbl-D status. We have previously found significant decreases in EGF levels in the CSF of 2-month TGX and 4-month TGX rats (Scalabrino et al., 1999), which do not correlate with the present findings showing that SC EGF levels remained unchanged in 2-month TGX rats but increased in 4-month TGX rats (Table 1). This discrepancy indicates that there is not necessarily any correspondence between SC and CSF EGF levels, possibly because CSF EGF comes at least partially from the blood as EGF freely crosses the blood-brain barrier in rodents (Scalabrino et al., 2004), and that the EGF synthesized in the SC of 4-month TGX rats seems to remain inside this part of the CNS rather than being secreted into CSF. We have previously observed partially repaired SC white matter (i.e. a simultaneous increase in the structured part and decrease in the vacuolated part) in untreated 4-month TGX rats (Scalabrino et al., 1997), which may be due to the increased SC EGF levels in 4-month TGX rats (Table 1). Only postoperative Cbl administration seems to restore a normal equilibrium between CSF and SC EGF levels in 4-month TGX, Cbl-treated rats, as it increases CSF (Scalabrino et al., 1999) and decreases SC EGF levels (Table 1). It is difficult to establish the role of abnormal EGFR expression in the pathogenesis of the hallmark intramyelinic edema in Cbl-D SC white matter, which is most severe 2 months after TG and after 3 months of a Cbl-D diet (Scalabrino et al., 1990; Tredici et al., 1998). Although the decreased EGFR expression in the SC of 2-month TGX rats seems to lose its

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EGF-dependence, the postoperative administration of EGF has still a “curative” effect on SC myelin ultrastructure in 2month TGX and EGF-treated rats (Scalabrino et al., 2000). It is therefore conceivable that, together with other abnormalities, the abnormal relationship between decreased EGFR expression and unchanged EGF levels in the SC of 2-month TGX rats contributes to the pathogenesis of myelin damage in Cbl-D central neuropathy.

4.

Experimental procedures

4.1.

In vivo treatments

Non-inbred adult male albino Sprague–Dawley rats (Charles River Italia, Calco, Italy) underwent TG to induce Cbl deficiency (Scalabrino et al., 1990), with LPT rats being used as controls. After TG or laparotomy (LP) the weight loss of the 2-month TGX and that of 4-month TGX rats were 10 ± 5% and 20 ± 5%, respectively. After TG or LP the rats were fed parenterally for the first 4 to 5 days. Thereafter, both these groups of rats ate standard pellets. Some rats were chronically fed a Cbl-D diet (Scalabrino et al., 1990), which was substantially equivalent to that of the controls, except that it was Cbl free and contained a sulfonamide, succinylsulfathiazol (1 g %), to decrease the growth of intestinal bacteria (Scalabrino et al., 1990). UO rats were used as controls for the rats fed the Cbl-D diet. Cbl replacement treatment was given to the TGX rats weekly for the first 2 months after TG (scheme a), or during the third and fourth months after TG (i.e., after CNS abnormalities had already appeared). (scheme b) (Scalabrino et al., 1997). The procedures involving the animals and their care were conducted in conformity with our institutional guidelines and in compliance with international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, Dec. 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 86-23, 1985).

4.2.

Organ collection

The rats were killed by means of ether anesthesia, 2 or 4 months after TG or LP and 8 and 12 months of the Cbl-D diet, and their SCs and livers were quickly removed and frozen on dry ice. To avoid the interference of the hepatic EGFR circadian rhythm (Scheving, 2000), all of the rats were killed between 08.00 and 10.00 a.m.

4.3.

Assessment of Cbl-D status

Markers of Cbl-D status (Cbl, homocysteine and methylmalonic acid) were checked in the sera, SCs, and livers of the rats as previously described (Scalabrino et al., 1997) (data not shown).

4.4.

Total RNA extraction

Samples of the different frozen organs (nearly 120 mg for each) were homogenized at 4 °C in 1 ml TRIZOL (Invitrogen-Life Technologies, Carlsbad, CA), and the RNA was extracted as previously described (Magnaghi et al., 2006).

28 4.5.

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Complementary EGFR mRNA probe and EGFR RPA

We generated a specific plasmid containing an insert of 508 bp coding for EGFR mRNA (NCBI Reference Sequence: NM_031507.1) into a pCR®II-TOPO® vector (Invitrogen). A complementary mRNA probe, corresponding to nucleotides 1033-1541, was generated by in vitro transcription with T7 polymerase (Promega, Madison, WI) in the presence of 12.5 μM 32 P-cytidine 5'-triphosphate (Amersham, Amersham, U.K.) (Magnaghi et al., 2006). The commercial pTRI-18S-rat plasmid (Ambion, Austin, TX) yielded a complementary rRNA probe that was used for internal housekeeping purposes. The assay was set up using total RNA from normal rat liver, which expresses high EGFR levels (Carver et al., 1996). Twenty micrograms of each RNA sample of SC and liver were dissolved in 20 μL of a hybridization solution (Magnaghi et al., 2006), and then, 150,000 cpm of the 32P-labeled EGFR probe and 50,000 cpm of the 32P-labeled 18S probe were added. The samples were incubated at 45 °C overnight, treated with 200 μL of digestion buffer solution (300 mM NaCl, 10 mM Tris–HCl pH 7.4, 5 mM EDTA pH 7.4), and an RNase cocktail (1 μg/μL RNase A and 20 U/μL RNase T1; Ambion), and then incubated for 15 min at 37 °C with 10 μg proteinase K (10 μg/μl) and 10 μL sodium dodecyl sulfate (from a 20% stock solution). The samples were phenol–chloroform extracted, precipitated, resuspended in loading buffer solution, and separated on a 5% polyacrylamide gel at 1800 V for 3 hrs (Magnaghi et al., 2006). Protected RNA fragments were visualized by means of autoradiography. Both the EGFR mRNA and 18S rRNA levels were assessed using densitometric analysis, and EGFR mRNA levels were quantified after normalization with 18S rRNA levels. The mean value of each control group was set at 100.

Applied Biosystems (Rn00580398_m1), which also supplied the primers and MGB probe annealing 18S rRNA used as the internal control. The primers were unlabeled, but the MGB probes were labeled at their 5′ end by a reporter dye (6carboxy-fluorescein for EGFR and 2′-chloro-7′-phenyl-1,4dichloro-6-carboxyfluorescein for 18S) and at their 3′ end by a non-fluorescent quencer dye. The primers and probe for rat EGFR were generated against different sites of mRNA with respect to the complementary EGFR mRNA probe used for RPA to improve the amplification efficiency of the rt-PCR. The primers and probe for rat EGFR used for rt-PCR and the complementary EGFR mRNA probe used for RPA map at the 5′-terminal sequence (external domain), where the region fulllength and truncated forms of EGFR are completely homologous (Petch et al., 1990). Duplex rt-PCR reactions were carried out to detect EGFR and 18S simultaneously in the same well. 50 ng cDNA was incubated with 25 μl TaqMan Universal PCR Master Mix, containing the Taq DNA polymerases (Applied Biosystems), 2.5 μl EGFR primer/probe mixture (Applied Biosystems), and 2.5 μl 18S primer/probe mixture. The fluorescence data were processed and analyzed using ABI PRISM sequence detection software (version 2.2.1.), and the measured fluorescence was plotted against the cycle number to determine the cycle threshold (Ct). The PCR amplification of EGFR mRNA was as efficient as that of 18S (data not shown). Relative EGFR mRNA expression is described using the ΔΔCt method (Livak and Schmittgen, 2001): ΔCt was obtained by subtracting the 18S Ct from the EGFR Ct in each sample; the mean values of EGFR mRNA expression in the controls within each experiment was set to 100, and therefore those of the Cbl-D organs were calculated as 100 × 2−ΔΔCt.

4.8. 4.6.

Five hundred nanograms of RNA from each tissue sample were reverse transcribed in 25 μl of a buffer containing: 500 μM deoxyribonucleoside triphosphated mixture (Applied Biosystem, Foster City, CA), 2.5 μM random hexamers (Applied Biosystem), 0.4 U/μl RNase inhibitor (Applied Biosystem), 5.5 mM MgCl2 (Applied Biosystem), and 3.12 U/μl recombinant Moloney murine leukemia virus reverse transcriptase (Applied Biosystem). The cycling conditions included incubation of this buffer at 25 °C for 10 min to maximize random hexamersRNA binding, followed by reverse transcription at 37 °C for 60 min and then inactivation at 95 °C for 10 min. The cDNA was synthesized and used for rt-PCR assay (see below).

4.7.

SC EGFR immunohistochemistry

cDNA synthesis

rt-PCR assay of EGFR mRNA

Thermus aquaticus(Taq)Man rt-PCR was carried out using a thermostable DNA polymerase with 5′–3′ endonucleolytic activity and a thermal cycler coupled to a fluorescence detection system (ABI PRISM 7900HT sequence detection system, Applied Biosystems). The cycling conditions used to optimize the amplification profile included denaturation at 95 °C for 10 min in order to activate DNA polymerase, followed by 40 15s-amplification cycles at 95 °C, and then annealing and extension (both at 60 °C for 1 min). The primers and minor groove binder (MGB) probe for rat EGFR were purchased from

Three LPT rats, three 2-month TGX rats, three 2-month TGX-, Cbl-treated rats and 3 rats fed a Cbl-D diet for 8 months were perfused as previously described (Mutti et al., 2007), and their SCs were processed (Mutti et al., 2007). Three coronal 10-μm serial sections from each SC segment of each rat were cut, collected on poly-L-lysine coated slides and treated with EDTA (pH 8.0) for 10 min in order to retrieve the antigen, and then incubated overnight at 4 °C with EGFR polyclonal rabbit antibody (Cell Signalling, Boston, MA) (diluted 1:100 weight/ volume (w/v) in sterile pyrogen-free saline). The immunocomplex was visualized using the ABC peroxidase method (Vector Laboratories, Burlingame, CA). The images were obtained using an optical microscope (Olympus, Tokyo, Japan) equipped with a digital CCD camera (C8510, Hamamatsu, Shizuoka, Japan) at 5× magnification, and analyzed using a computerized image analysis system (Photoshop CS2, Adobe, San Jose, CA). For immunostaining quantification, 12 different ROI (3200 μm2 each) in the white matter of the SC posterior portion (6 in its right part and 6 in its left part) were chosen randomly for each of the three sections of each SC segment of each rat. The SC posterior portion of Cbl-D rat was chosen because the myelin of the cuneate fasciculus and gracile fasciculus shows the hallmarks of Cbl-deficiency-induced lesions (i.e. spongy vacuolation and intramyelinic edema) and the rest (i.e. dorsal corticospinal tract) is morphologically and ultrastructurally unaffected (Scalabrino et al., 2000;

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Tredici et al., 1998). The immunohistochemistry staining was quantitatively analyzed using the automated image analysis software Definiens Tissue Studio (Definiens Inc, Parsippany, NJ). All of the examinations were made blindly: i.e. without knowing to which experimental group the animals belonged. The homoscedasticity of the variances of the data belonging to the four groups was tested using Bartlett's test (Bartlett, 1937), the results of which always gave chi-square values showing that the variances were not significantly different. ANOVA was used to determine the statistical significance of the differences in the means of the different groups, and the statistical significance of the differences between each pair of means was evaluated using Tukey's test. An α-level of 0.05 or less was fixed as the limit of statistical significance for all of the tests.

4.9.

EGF assay

EGF concentrations were determined in pooled SC samples (a total of nearly 0.9 g) from each group of rats and whole liver samples from each rat. The samples were homogenized in ice-cold 10× Dulbecco's phosphate buffer (without Ca2+ and Mg2+), diluted 1:2 w/v for SC, and 1:1 w/v for liver. After centrifugation, the supernatants of the SCs and livers were evaporated overnight in a vortex evaporator (Buchler Instruments, New York, NY), and the EGF levels of each salinediluted sample were determined using a commercially available ELISA kit (Prepotech, London, U.K.). The optical densitometry (OD) data were analyzed by means of KCjunior curve-fitting software.

4.10.

Protein determination

Protein levels were determined using Lowry's method.

Acknowledgments E.M. and V.M. are deeply indebted to Dr. H. Shelton Earp, III (University of North Carolina, Chapel Hill, NC) for his precious help in setting up the EGFR plasmid, and to Dr. Ileana Zucchi (C.N.R., Milan, Italy) for her help in setting up the EGFR primer for RPA. E.M. and D.V. thank Dr. M. A. Pierotti, Dr. Maria Grazia Daidone, and Dr. D. Conte (Istituto Nazionale dei Tumori, Milan, Italy) for allowing us to use their thermal cycler ABI PRISM 7900 HT. G.S. would like to thank Dr. F. Pamparana (Bayer Italia, Milan, Italy) for his financial help, and Pfizer Italia (Rome, Italy) and Sanofi-Aventis S.p.A. (Milan, Italy) for their kind supply of antibiotics. S.P. and P.P. D.F. would all like to thank Dr. G. Mazzarol (Department of Medicine, Surgery, and Dentistry, University of Milan, IFOM-IEO Campus, Milan, Italy) for his histopathological analysis of the SC EGFR immunohistochemical staining, Dr. M. Abbate (Immagini & Computer, Bareggio, Italy) for kindly providing the automated image analysis software, Dr. D. Parazzoli (Department of Medicine, Surgery, and Dentistry, University of Milan, IFOM-IEO Campus, Milan, Italy) for his technical assistance in image acquisition and processing, and Dr. G. D'Ario (Department of Medicine, Surgery, and Dentistry, University of Milan, IFOMIEO Campus, Milan, Italy) for the statistical analysis. Finally,

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we would all also like to thank Mr. P. Lamanna (“Città Studi” Department, University of Milan) for his technical assistance in in vivo treatments.

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