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Expression of mRNAs for Lysyl Oxidase and Type III Procollagen in Cultured Fibroblasts from Patients with the Menkes and Occipital Horn Syndromes as Determined by Quantitative Polymerase Chain Reaction
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 328, No. 1, April 1, pp. 101–106, 1996 Article No. 0148
Expression of mRNAs for Lysyl Oxidase and Type III Procollagen in Cultured Fibroblasts from Patients with the Menkes and Occipital Horn Syndromes as Determined by Quantitative Polymerase Chain Reaction R. Kemppainen,* E.-R. Ha¨ma¨la¨inen,* H. Kuivaniemi,† G. Tromp,† T. Pihlajaniemi,* and K. I. Kivirikko*,1 *The Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Kajaanintie 52 A, FIN-90220 Oulu, Finland; and †Department of Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received September 7, 1995, and in revised form January 11, 1996
The Menkes syndrome and the occipital horn syndrome are two X-linked recessively inherited disorders characterized by abnormalities in copper metabolism. These abnormalities are associated with a reduction in the activity of lysyl oxidase (EC 1.4.3.13), an extracellular copper enzyme that initiates the crosslinking of collagens and elastin. We report here that the amount of lysyl oxidase mRNA, as studied by Northern blotting, and the number of lysyl oxidase mRNA molecules per picogram of RNA, as determined by a quantitative PCR method, were decreased in three cultured skin fibroblast lines from patients with the Menkes syndrome and two from patients with the occipital horn syndrome compared with four control cell lines. The decreased lysyl oxidase activity found in these disorders thus appears to be at least in part due to a pretranslational mechanism. No decrease was found in the number of the b-actin mRNA molecules in the Menkes cell lines, but rather a slight increase, whereas a decrease was found in these molecules in the occipital horn cell lines. An additional abnormality found in the Menkes cell lines was a significant increase in the number of mRNA molecules for type III procollagen in two of the three cell lines investigated. The present and previous data indicate that the Menkes syndrome may involve several abnormalities in the expression of genes for connective tissue proteins. q 1996 Academic Press, Inc. Key Words: lysyl oxidase; type III procollagen; quantitative PCR.
1 To whom correspondence should be addressed. Fax: Intl-358-81537 5810.
The Menkes syndrome and the occipital horn syndrome are two X-linked recessively inherited disorders with alterations in copper metabolism and extensive connective tissue abnormalities. The occipital horn syndrome is characterized by bladder diverticula with spontaneous ruptures, inguinal hernias, slight skin laxity, and hyperextensibility, and a number of skeletal changes, a peculiar feature being occipital horn-like exostoses. The Menkes syndrome also includes bladder diverticula, mildly increased skin laxity and hyperextensibility and skeletal abnormalities, but unlike the occipital horn syndrome, it also involves neurologic degeneration, mental retardation and vascular complications so severe that the disease is usually lethal by the age of 3 years (for reviews, see 1–3). A similar array of abnormalities is found in the X-linked mottled series of alleic mutant mice (2). All these diseases appear to be due to mutations in an X-chromosomal gene coding for a copper-transporting P-type ATPase (4–7). As a consequence of these mutations, serum copper and ceruloplasmin concentrations are low, whereas cultured fibroblasts and many other cells have markedly elevated copper concentrations (1, 2). All the connective tissue manifestations found in these disorders are probably due to a reduction in the activity of lysyl oxidase, an extracellular copper enzyme that initiates the crosslinking of collagens and elastin by catalyzing oxidative deamination of the eamino group in certain lysine and hydroxylysine residues (8, 9). A deficiency in this enzyme activity has been demonstrated in skin specimens (10, 11) and in the medium of cultured fibroblasts (10–13) from patients with the occipital horn syndrome and in the me101
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dium of cultured fibroblasts from patients with the Menkes syndrome (11, 13–15), but the mechanisms leading to the deficient enzyme activity are unknown. Cultured skin fibroblasts from patients with these disorders have been found to contain and secrete reduced amounts of lysyl oxidase protein (13, 16), which suggests that either the copper deficiency inhibits the transcription or translation of lysyl oxidase or a copperdeficient enzyme protein is rapidly degraded after its synthesis. Lysyl oxidase cDNA clones have been isolated from rat (17, 18), human (19, 20), and chick (21) cDNA libraries, and the complete exon–intron organization has been determined for the human (19) and the mouse (23) lysyl oxidase genes. The human gene is located in the region q23.3–q31.2 of chromosome 5 (19), which clearly excludes primary defects in lysyl oxidase in these X-linked disorders. The availability of cDNA probes for human lysyl oxidase made it possible for us to study further the mechanisms involved in the lysyl oxidase deficiency in the Menkes syndrome and the occipital horn syndrome by measuring the levels of the mRNAs for this enzyme in cultured fibroblasts from patients with these disorders. In addition, the levels of mRNA for type III procollagen were measured. While the work was in progress, two laboratories reported contradictory data on lysyl oxidase mRNA levels in the Menkes syndrome obtained by Northern analysis, one reporting a low mRNA level in one patient (16), whereas the other found no change in lysyl oxidase mRNA in three patients (24). We therefore paid special attention to accurate measurements by using a quantitative polymerase chain reaction (PCR) technique. MATERIALS AND METHODS Cell cultures. Three Menkes fibroblast cell lines (GMO1057, GMO0220, GMO3700) were obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). The two occipital horn cell lines were described earlier (12). Four control fibroblast cultures were established from skin specimens from three healthy subjects and one fetus. The cells were grown in 65-cm2 plastic tissue culture dishes in 10 ml of Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 100 mg/ml L-glutamine. For the measurement of lysyl oxidase activity fibroblasts that had just reached confluence were washed twice in 0.14 M NaCl, 0.1 M sodium phosphate, pH 7.4, and were then cultured for 24 h in 5 ml of the above medium without serum but supplemented with 5 mg/ml bovine serum albumin. Northern blot analysis. RNA was electrophoresed in a 0.8% (w/v) agarose gel containing 2 M formaldehyde and was transferred to a nitrocellulose filter (25). The Northern filter was hybridized to a 2 ng/ml solution of a 32P-labeled human lysyl oxidase cDNA clone, HLO20 (19), and a 1200-bp clone that codes for glyceraldehyde-3phosphate dehydrogenase (American Type Culture Collection, probe 57091) in 50% (v/v) formamide, 51 SSC2 (11 SSC Å 0.15 M NaCl, 0.015 M sodium citrate, pH 6.8), denatured salmon sperm DNA (0.25
2
Abbreviation used: SSC, standard saline citrate.
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mg/ml), 0.1% SDS, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin for 24 h at 417C. The filters were washed twice for 30 min in 0.51 SSC, 0.05% SDS at room temperature and once for 15 min at 657C. RNA preparation. Control constructs were prepared by PCR using lysyl oxidase cDNA (19), b-actin cDNA (G. Tromp and H. Kuivaniemi, unpublished results), and type III procollagen cDNA (26) clones as templates as described (27). Four primers derived from the cDNA sequences of lysyl oxidase were designed: sense primer LO-1, 5*-CGC GAA TTC GCC GGG AGA AGT TCC TGC TCT C-3* (covering nucleotides 671–692); antisense primer LO-2, 5*-CGC GAA TTC CTG TGG TAG CCA TAG TCA CAG G-3 * (covering nucleotides 1216–1238); antisense primer LO-3, 5*-CGC AAG CTT GGG AGA CCG TAC TGG AAG TAG CC-3* (covering nucleotides 823–846); and sense primer LO-4, 5*-CGC AAG CTT CCT GAG ATG CGC GGC GGA GG-3* (covering nucleotides 954–973); and also four primers derived from the cDNA sequences of b-actin: sense primer b-actin1, 5*-CGC GAA TTC GTG AAC TTT GGG GGA TGC TC-3* (covering nucleotides 1025–1044); antisense primer b-actin-2, 5*-CGC GAA TTC GTG AAC TTT GGG GGA TGC TC-3* (covering nucleotides 1396–1416); antisense primer b-actin-3, 5*-CGC AAG CTT CTG CTT GCT GAT CCA CAT CTG-3* (covering nucleotides 1147–1168); and sense primer b-actin-4, 5*-CGC AAG CTT AGT TGC GTT ACA CCC TTT CTT G-3* (covering nucleotides 1229–1250); and two primers derived from the cDNA sequences of the proa1 chain of type III procollagen: sense primer COL3A1-1, 5*-CGG AAT TCT TGG GAT TGC TGG GAT CAC T-3* (covering nucleotides 2934–2954) and antisense primer COL3A1-2, 5*-CGC GAA TTC GTG AAC TTT GGG GGA TGC TC-3* (covering nucleotides 3844–3866). The primers numbered 1 and 2 in each case contained a site for the EcoRI restriction endonuclease, and primers numbered 3 and 4 a site for HindIII. The oligonucleotide primers were synthesized in an Applied Biosystems DNA synthesizer (Department of Biochemistry, University of Oulu). Two PCRs were first carried out for the lysyl oxidase and bactin cDNAs, one with primers 1 and 3 and the other with primers 2 and 4. PCR was performed at a final concentration of 11 PCR buffer (10 mM Tris–HCl, pH 8.8, 1.5 mM MgCl2 , 50 mM KCl, 0.1% Triton X-100) (Dynazyme), 200 mM dNTPs, 10 pmol of each primer, 1 unit of thermostable DNA polymerase (Dynazyme) in a total volume of 50 ml. The amplification profile involved denaturation at 947C for 1 min 30 s, primer annealing at 54–647C for 1 min, and extension at 727C for 1 min. The amplified products were digested with HindIII (Pharmacia) and then ligated at the HindIII sites. The ligated fragment was then used as a template in a PCR with primers 1 and 2. The product now contained a deletion. In the amplification reaction for the type III procollagen cDNA only primers 1 and 2 were used and the deletion was generated by digesting the PCR product with TaqI restriction enzyme (Pharmacia), thereby removing a small part from the center of the PCR product. The PCR products with deletions were subcloned into plasmid Bluescript SK (Stratagene) as described (25). The plasmids were purified by the CsCl method (25). These plasmids were used as templates for transcription by the T3 or T7 polymerase according to the transcription protocol of Ambion, Inc. The resulting control RNA was quantified by two methods: absorbance at 260 nm and addition of 5 mCi of [32P]UTP (400 Ci/mmol, Amersham) to the in vitro transcription reaction and determination of its incorporation by trichloroacetic acid precipitation as advised by Ambion, Inc. Total cellular RNA was isolated from the cells by the method of acid guanidinium thiocyanate–phenol–chloroform (28). cDNA synthesis and PCR amplification. A 30-ml reverse transcription reaction was carried out on 0.5–6 mg of total cellular RNA and an in vitro-synthesized control containing 7.5 1 108 molecules of RNA for type III procollagen, 11.1 1 108 molecules for lysyl oxidase, or 5.5 1 109 molecules for b-actin. The reaction mixture contained 11 first-strand buffer (50 mM Tris–HCl, pH 8.3, 5 mM KCl, 3 mM MgCl2) (Gibco BRL), 10 mM dithiothreitol, 0.5 mM dNTP, 4 pmol random primers (Gibco BRL), and 100 units of Moloney murine leu-
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LYSYL OXIDASE mRNA IN HUMAN MENKES AND OCCIPITAL HORN SYNDROME CELLS TABLE I
Lysyl Oxidase Activity and Number of mRNA Molecules per Picrogram of Total RNA for Lysyl Oxidase mRNA Produced by the Various Cell Lines
Cell line Control cells Embryonal skin fibroblasts HES Adult skin fibroblasts 9011 9505 20009 Group mean Menkes cells GMO 1057 GMO 220 GMO 3700 Group mean Occipital horn cells ED58 ED 3999 Group mean
Lysyl oxidase activity (dpm/106 cells)
2130
Number of lysyl oxidase mRNA molecules/ pg total RNAa
440 { 25b (4)c
1780 1100 1430 1640 { 420
430 360 340 390
{ { { {
120 90 50 90
(13) (6) (13) (36)
790 590 670 680 { 80
290 230 150 230
{ { { {
160 40 20 110
(11) (10) (8) (29)
630 400 520 { 100
290 { 110 (8) 190 { 30 (6) 240 { 100 (14)
a
The values of the control cells are the same as in Ref. 27. SD. c Number of values used in calculating the mean. b
kemia virus reverse transcriptase (Gibco BRL), and was incubated at 377C for 60 min. PCR was performed in a final concentration of 11 PCR buffer, 160 mM dNTPs, 10 pmol of each 3* primer, 10 pmol of each 32P end-labeled 5* primer, and 1 unit of thermostable DNA polymerase in a total volume of 50 ml. The amplification involved denaturation at 947C for 1 min 30 s, annealing at 607C for 1 min 20 s, and extension at 727C for 1 min 30 s. The oligonucleotides were labeled with T4 polynucleotide kinase and [g32P]ATP (3000 Ci/mmol, Amersham Corp.). The sizes of the PCR products were 568 and 508 bp for lysyl oxidase, 392 and 337 bp for b-actin, and 919 and 748 bp for type III procollagen. Quantitative analysis. A 10-ml aliquot of each PCR reaction mixture was electrophoresed on 6% (v/v) polyacrylamide gels. The gels were dried and autoradiographed on X-ray films (Kodak). Appropriate bands were cut out from the gel and radioactivity was determined with a liquid scintillation counter. The efficiency of amplification was determined using the formula log Y Å log A / n log(1 / R), where Y is the extent of amplification, A is the amount of initial material, R is the efficiency, and n is the number of cycles. It is possible to estimate Y with reasonable accuracy by measuring the extent of incorporation of 32P-labeled primer into the PCR product, and R can be deduced from the slope of the semilog plot (29, 30). The Statistica software package (StatSoft), specifically the Spjøtvoll and Stoline extension of the t test, was used for comparisons between cell lines and groups of cell lines. The test is a powerful, i.e., conservative, test that can be used for simultaneous comparison of multiple groups of unequal n (31). Lysyl oxidase activity assay. L-[4,6-3H]lysine-labeled purified chick embryo elastin substrate was prepared (32), and the lysyl oxidase activity of the cell culture media was measured with 125,000 cpm of elastin substrate in a total volume of 0.8 ml (32). The samples were incubated for 10 h at 377C, and the tritiated water formed
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during the incubation was isolated by vacuum distillation and quantified with a liquid scintillation counter (32).
RESULTS
Lysyl Oxidase Activity in the Medium of Cultured Fibroblasts In agreement with previous data (10, 12–15), a decreased level of lysyl oxidase activity was seen in the culture medium of all three Menkes cell lines and the two occipital horn cell lines (Table I). The enzyme activity in the Menkes cell lines ranged from 35 to 49% of the control mean and that of the two occipital horn cell lines from 23 to 37%. The mean enzyme activity in both groups was significantly below the control mean (P Å 0.006). Northern Analysis Human lysyl oxidase mRNA is found by Northern analysis in multiple forms with approximate sizes of about 5.5, 4.3 (major mRNA), 2.5, and 2.0 kb, although the 2.5- and 2.0-kb mRNAs are usually not resolved in short runs (22, 33). No qualitative differences were found in this pattern of mRNAs in the Menkes and occipital horn fibroblasts, but the quantity of all the mRNA species appeared to be decreased relative to the control cell lines (Fig. 1). When the same blot was hybridized to a probe for glyceraldehyde-3-phosphate dehydrogenase, no significant differences were seen between the cell lines (not shown). Quantitative Analysis and Internal Standard To obtain a more accurate and reliable measurement of the lysyl oxidase mRNA levels, a quantitative PCR method was used in which control RNAs for lysyl oxidase, b-actin, and type III procollagen, each containing a small deletion compared with the full-length cDNA
FIG. 1. Northern blot using lysyl oxidase cDNA probe. Total RNA from the cultured control skin fibroblasts (9011 and HES), fibroblasts of one occipital horn syndrome patient (ED58), and fibroblasts of two Menkes syndrome patients (GMO 3700 and GMO 220) was isolated, electrophoresed on a 1% agarose gel, and transferred onto membrane that was probed with a 32P-labeled lysyl oxidase cDNA probe, as described under Materials and Methods. The two main lysyl oxidase mRNA species of about 5.5 and 4.3 kb are shown.
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FIG. 2. Autoradiogram of the cRNA and target mRNA products for type III procollagen, lysyl oxidase, and b-actin. The cRNA and target mRNA products for type III procollagen, lysyl oxidase, and b-actin were separated by 6% polyacrylamide gel electrophoresis. Serial 1:2 dilutions of the cDNA mixture were amplified using specific oligonucleotides. One of the primers was 32P-end-labeled.
clones, were synthesized from plasmids. The deletion was made by PCR in the case of lysyl oxidase and bactin cDNA or by enzymatic digestion in the case of type III procollagen cDNA. First-strand cDNA was synthesized in the same tube for a mixture of control RNA and RNA isolated from the cell line. Dilution series of the cDNA mixtures were then coamplified in the same tube, and the reactions were terminated in the exponential phase of amplification. These control RNAs served as internal standards for the reverse transcription reactions and PCRs and were used to generate standard curves for quantifying specific target mRNAs from experimental samples. The PCR primers used were the same for the control RNAs and target mRNAs, and thus there were no primer efficiency differences between these templates. In fact, determination of the amplification efficiencies failed to distinguish between the control construct and the cDNA from the cellular RNA for all three mRNAs studied. The size differences between the PCR products permitted easy separation of the cRNA product from the target mRNA product by gel electrophoresis (Fig. 2). The amounts of radioactivity recovered from the excised gel band were plotted against the template concentrations. The reaction rates of the control RNA and the target mRNA amplifications were identical in the exponential phase of the PCR reaction. This allowed construction of a standard curve for the control RNAs and extrapolation to a copy number for the target mRNAs isolated from cultured cells. Quantification of the Lysyl Oxidase, b-Actin, and Type III Procollagen mRNAs in the Various Cell Types The numbers of lysyl oxidase mRNA molecules in the four control cell lines were very similar, ranging from
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about 340 molecules/pg total RNA in cell line 20009 to 440 molecules/pg in the HES cells (Table I). The mean value for the three Menkes cell lines studied was significantly lower (P Å 0.0001), about 60% of the control mean with the values ranging from about 150 molecules/pg RNA in the GMO 3700 cells to 290 molecules/ pg in the GMO 1057 cells (Table I). The mean for the two occipital horn cell lines was likewise about 60% of the control mean (P Å 0.001), the values being 190 and 290 molecules/pg. Thus the values in all three Menkes cell lines and two occipital horn cell lines were below that in the lowest control. The number of b-actin mRNA molecules per picogram of total RNA showed a larger variation among the four control lines (Table II). A slightly higher mean was found in the group of the Menkes cells, whereas the mean for the two occipital horn cell lines was decreased (P Å 0.0001). The lowest value among all the cell lines, about 2000 molecules/pg RNA, was nevertheless found in the control cell line 9011 (Table II). The number of mRNA molecules encoding type III procollagen (Table II) was very high, about 230% of the mean of the controls, in two of the three Menkes cell lines (GMO 220, P Å 0.0002; and GMO 3700, P Å 0.0094), whereas this number was not increased in the third line (GMO 1057, P Å 0.816). No increase was found in this value in the only occipital horn cell line
TABLE II
Number of mRNA Molecules per Picogram of Total RNA for b-Actin and Type III Procollagen in the Various Cell Lines
Cell line Control cells Embryonal skin fibroblasts HES Adult skin fibroblasts 9011 9505 20009 Group mean Menkes cells GMO 1057 GMO 220 GMO 3700 Group mean Occipital horn cells ED 58 ED 3999 Group mean a
Number of type Number of b-actin III procollagen mRNA molecules/ mRNA molecules/ pg total RNA pg total RNAa
5170 { 114b (7)c
380 { 20 (6)
2000 6600 5500 4500
{ { { {
400 1690 1600 2230
(9) (7) (3) (26)
470 670 980 590
120 120 500 310
(11) (7) (6) (30)
5250 4930 4230 4780
{ { { {
1530 430 1090 1110
(6) 460 { 50 (7) 1380 { 220 (7) 1350 { 160 (20) 1050 { 480
(6) (8) (3) (17)
2400 { 400 (9) 3400 { 530 (5) 2820 { 630 (13)
{ { { {
390 { 70 (7) 390 { 70 (7)
The values of the control cells are the same as in Ref. 27. SD. c Number of values used in calculating the mean. b
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LYSYL OXIDASE mRNA IN HUMAN MENKES AND OCCIPITAL HORN SYNDROME CELLS
analyzed, where the number was about 65% of the control mean and only slightly above that in the lowest control (Table II). DISCUSSION
The present data indicate that the numbers of lysyl oxidase mRNA molecules per picogram RNA were significantly decreased in the three cultured skin fibroblast lines from patients with the Menkes syndrome and the two from patients with the occipital horn syndrome. This effect was specific in the Menkes cell lines, in that no decrease was found in the number of b-actin mRNA molecules but rather a slight increase, whereas the cells from patients with the occipital horn syndrome also showed a decrease in the number of the bactin mRNA molecules. The data thus indicate that the decreases seen in the amounts of lysyl oxidase activity in these two diseases are not simply due to abnormal utilization of copper as a cofactor for the enzyme but are at least in part due to a pretranslational mechanism, either diminished transcription of the lysyl oxidase gene or impaired processing or stability of the mRNA. The decrease in the number of the mRNA molecules was not as marked as that in the enzyme activity, however, suggesting that additional mechanisms contribute to the low enzyme activity. One such mechanism might well be a poorer stability of the copperdeficient lysyl oxidase protein, as earlier studies have indicated a large decrease in the amount of this enzyme in cells from patients with the Menkes syndrome (13, 16) and occipital horn syndrome (13), and from the tortoiseshell mouse (16), a murine analogue of the Menkes syndrome (2). The present data suggesting a diminished number of lysyl oxidase mRNA molecules agree with figures recently reported in cells from one Menkes patient and one tortoiseshell mouse (16), except that the magnitudes of the decreases obtained on that occasion, using only Northern blotting, were much larger than those determined here. The data disagree with a recent report by another group (24), however, who found no decrease in the amount of the lysyl oxidase mRNA in cells from three Menkes patients by Northern blotting. Because the number of lysyl oxidase mRNA molecules in one of our Menkes cell lines was only about 15% below that in the lowest control, it is evident that the decreases in cells from some patients cannot be detected by Northern blotting. Furthermore, it seems likely that some Menkes cell lines may have lysyl oxidase mRNA molecule numbers that are within the control range. It is now well established that the basic defect in the Menkes syndrome and the occipital horn syndrome is a mutation in an X-chromosomal gene encoding a coppertransporting ATPase (4–7). The present and previous
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data (16) indicate that the consequences of this defect extend far beyond those which could be expected on the basis of the simple alteration in the availability of copper to the enzymes that utilize this cation, such as lysyl oxidase. A previous study has reported an increased level of mRNA for the proa1 chain of type I procollagen in cells from a Menkes patient and a tortoiseshell mouse (16). The present data extend this finding by indicating that the number of mRNA molecules for type III procollagen was increased in two of the three Menkes cell lines studied. Accordingly, the ratio of lysyl oxidase mRNA molecules to type III procollagen mRNA molecules in these two cell lines was only about 25% (GMO 220) or 17% (GMO 3700) of that in the controls. The amount of elastin mRNA has also been reported to be markedly altered in cells from a Menkes patient and a tortoiseshell mouse (16), but the change is opposite in direction to those seen in the amounts of type I procollagen (16) and type III procollagen mRNAs, in that the amount of elastin mRNA was markedly decreased. An additional abnormality found in the present study was that the number of b-actin mRNA molecules was decreased in cells from patients with the occipital horn syndrome. The mechanisms that lead to these changes in the various mRNA levels in the Menkes and occipital horn syndrome cells are currently unknown. Analysis of additional Menkes cell lines will allow the use of statistical tests to determine whether cell lines from patients diagnosed with the Menkes syndrome can be classified into two or more categories. Should multiple categories exist, it is possible that variance in the levels of expression of some of the mRNAs for extracellular matrix proteins could explain some or all of the phenotypic variance. ACKNOWLEDGMENTS We are grateful to Aira Harju and Riitta Poloja¨rvi for their technical assistance. The work was supported by grants from the Medical Research Council of the Academy of Finland, the Sigrid Juse´lius Foundation and the Paulo Foundation.
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28. 29. 30. 31. 32. 33.
Shows, T. B., Boyd, C. D., and Deak, S. B. (1992) Matrix 12, 242–248. Wu, Y., Rich, C. B., Lincecum, J., Trackman, P. C., Kagan, H. M., and Foster, J. A. (1992) J. Biol. Chem. 267, 24199–24206. Ha¨ma¨la¨inen, E.-R., Kemppainen, R., Pihlajaniemi, T., and Kivirikko, K. I. (1993) Genomics 17, 544–548. Contente, S., Csiszar, K., Kenyon, K., and Friedman, R. M. (1993) Genomics 16, 395–400. Yeowell, H. N., Marshall, M. K., Walker, L. C., Han, V., and Pinnell, S. R. (1994) Arch. Biochem. Biophys. 308, 299–305. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Tromp, G., Kuivaniemi, H., Shikata, H., and Prockop, D. J. (1989) J. Biol. Chem. 264, 1349–1352. Ha¨ma¨la¨inen, E. R., Kemppainen, R., Kuivaniemi, H., Tromp, G., Vaheri, A., Pihlajaniemi, T., and Kivirikko, K. I. (1995) J. Biol. Chem. 270, 21590–21593. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156– 159. Chelly, J., Montanas, D., Pinset, C., Berwad-Netter, Y., Kaplan, J.-C., and Kahn, A. (1990) Eur. J. Biochem. 187, 691–698. Wang, A. M., Doyle, M. V., and Mark, D. F. (1989) Proc. Natl. Acad. Sci. USA 86, 9717–9721. Spjøtvoll, E., and Stoline, M. R. (1973) J. Am. Stat. Assoc. 68, 975–978. Kagan, H. M., and Sullivan, K. A. (1982) Methods Enzymol. 82, 637–650. Svinarich, D. M., Twomey, T. A., Macauley, S. P., Krebs, C. J., Yang, T. P., and Krawetz, S. A. (1992) J. Biol. Chem. 267, 14382– 14387.
AP: Archives
Vol. 328, No. 1, April 1, pp. 101–106, 1996 Article No. 0148
Expression of mRNAs for Lysyl Oxidase and Type III Procollagen in Cultured Fibroblasts from Patients with the Menkes and Occipital Horn Syndromes as Determined by Quantitative Polymerase Chain Reaction R. Kemppainen,* E.-R. Ha¨ma¨la¨inen,* H. Kuivaniemi,† G. Tromp,† T. Pihlajaniemi,* and K. I. Kivirikko*,1 *The Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Kajaanintie 52 A, FIN-90220 Oulu, Finland; and †Department of Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received September 7, 1995, and in revised form January 11, 1996
The Menkes syndrome and the occipital horn syndrome are two X-linked recessively inherited disorders characterized by abnormalities in copper metabolism. These abnormalities are associated with a reduction in the activity of lysyl oxidase (EC 1.4.3.13), an extracellular copper enzyme that initiates the crosslinking of collagens and elastin. We report here that the amount of lysyl oxidase mRNA, as studied by Northern blotting, and the number of lysyl oxidase mRNA molecules per picogram of RNA, as determined by a quantitative PCR method, were decreased in three cultured skin fibroblast lines from patients with the Menkes syndrome and two from patients with the occipital horn syndrome compared with four control cell lines. The decreased lysyl oxidase activity found in these disorders thus appears to be at least in part due to a pretranslational mechanism. No decrease was found in the number of the b-actin mRNA molecules in the Menkes cell lines, but rather a slight increase, whereas a decrease was found in these molecules in the occipital horn cell lines. An additional abnormality found in the Menkes cell lines was a significant increase in the number of mRNA molecules for type III procollagen in two of the three cell lines investigated. The present and previous data indicate that the Menkes syndrome may involve several abnormalities in the expression of genes for connective tissue proteins. q 1996 Academic Press, Inc. Key Words: lysyl oxidase; type III procollagen; quantitative PCR.
1 To whom correspondence should be addressed. Fax: Intl-358-81537 5810.
The Menkes syndrome and the occipital horn syndrome are two X-linked recessively inherited disorders with alterations in copper metabolism and extensive connective tissue abnormalities. The occipital horn syndrome is characterized by bladder diverticula with spontaneous ruptures, inguinal hernias, slight skin laxity, and hyperextensibility, and a number of skeletal changes, a peculiar feature being occipital horn-like exostoses. The Menkes syndrome also includes bladder diverticula, mildly increased skin laxity and hyperextensibility and skeletal abnormalities, but unlike the occipital horn syndrome, it also involves neurologic degeneration, mental retardation and vascular complications so severe that the disease is usually lethal by the age of 3 years (for reviews, see 1–3). A similar array of abnormalities is found in the X-linked mottled series of alleic mutant mice (2). All these diseases appear to be due to mutations in an X-chromosomal gene coding for a copper-transporting P-type ATPase (4–7). As a consequence of these mutations, serum copper and ceruloplasmin concentrations are low, whereas cultured fibroblasts and many other cells have markedly elevated copper concentrations (1, 2). All the connective tissue manifestations found in these disorders are probably due to a reduction in the activity of lysyl oxidase, an extracellular copper enzyme that initiates the crosslinking of collagens and elastin by catalyzing oxidative deamination of the eamino group in certain lysine and hydroxylysine residues (8, 9). A deficiency in this enzyme activity has been demonstrated in skin specimens (10, 11) and in the medium of cultured fibroblasts (10–13) from patients with the occipital horn syndrome and in the me101
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dium of cultured fibroblasts from patients with the Menkes syndrome (11, 13–15), but the mechanisms leading to the deficient enzyme activity are unknown. Cultured skin fibroblasts from patients with these disorders have been found to contain and secrete reduced amounts of lysyl oxidase protein (13, 16), which suggests that either the copper deficiency inhibits the transcription or translation of lysyl oxidase or a copperdeficient enzyme protein is rapidly degraded after its synthesis. Lysyl oxidase cDNA clones have been isolated from rat (17, 18), human (19, 20), and chick (21) cDNA libraries, and the complete exon–intron organization has been determined for the human (19) and the mouse (23) lysyl oxidase genes. The human gene is located in the region q23.3–q31.2 of chromosome 5 (19), which clearly excludes primary defects in lysyl oxidase in these X-linked disorders. The availability of cDNA probes for human lysyl oxidase made it possible for us to study further the mechanisms involved in the lysyl oxidase deficiency in the Menkes syndrome and the occipital horn syndrome by measuring the levels of the mRNAs for this enzyme in cultured fibroblasts from patients with these disorders. In addition, the levels of mRNA for type III procollagen were measured. While the work was in progress, two laboratories reported contradictory data on lysyl oxidase mRNA levels in the Menkes syndrome obtained by Northern analysis, one reporting a low mRNA level in one patient (16), whereas the other found no change in lysyl oxidase mRNA in three patients (24). We therefore paid special attention to accurate measurements by using a quantitative polymerase chain reaction (PCR) technique. MATERIALS AND METHODS Cell cultures. Three Menkes fibroblast cell lines (GMO1057, GMO0220, GMO3700) were obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). The two occipital horn cell lines were described earlier (12). Four control fibroblast cultures were established from skin specimens from three healthy subjects and one fetus. The cells were grown in 65-cm2 plastic tissue culture dishes in 10 ml of Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 100 mg/ml L-glutamine. For the measurement of lysyl oxidase activity fibroblasts that had just reached confluence were washed twice in 0.14 M NaCl, 0.1 M sodium phosphate, pH 7.4, and were then cultured for 24 h in 5 ml of the above medium without serum but supplemented with 5 mg/ml bovine serum albumin. Northern blot analysis. RNA was electrophoresed in a 0.8% (w/v) agarose gel containing 2 M formaldehyde and was transferred to a nitrocellulose filter (25). The Northern filter was hybridized to a 2 ng/ml solution of a 32P-labeled human lysyl oxidase cDNA clone, HLO20 (19), and a 1200-bp clone that codes for glyceraldehyde-3phosphate dehydrogenase (American Type Culture Collection, probe 57091) in 50% (v/v) formamide, 51 SSC2 (11 SSC Å 0.15 M NaCl, 0.015 M sodium citrate, pH 6.8), denatured salmon sperm DNA (0.25
2
Abbreviation used: SSC, standard saline citrate.
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mg/ml), 0.1% SDS, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin for 24 h at 417C. The filters were washed twice for 30 min in 0.51 SSC, 0.05% SDS at room temperature and once for 15 min at 657C. RNA preparation. Control constructs were prepared by PCR using lysyl oxidase cDNA (19), b-actin cDNA (G. Tromp and H. Kuivaniemi, unpublished results), and type III procollagen cDNA (26) clones as templates as described (27). Four primers derived from the cDNA sequences of lysyl oxidase were designed: sense primer LO-1, 5*-CGC GAA TTC GCC GGG AGA AGT TCC TGC TCT C-3* (covering nucleotides 671–692); antisense primer LO-2, 5*-CGC GAA TTC CTG TGG TAG CCA TAG TCA CAG G-3 * (covering nucleotides 1216–1238); antisense primer LO-3, 5*-CGC AAG CTT GGG AGA CCG TAC TGG AAG TAG CC-3* (covering nucleotides 823–846); and sense primer LO-4, 5*-CGC AAG CTT CCT GAG ATG CGC GGC GGA GG-3* (covering nucleotides 954–973); and also four primers derived from the cDNA sequences of b-actin: sense primer b-actin1, 5*-CGC GAA TTC GTG AAC TTT GGG GGA TGC TC-3* (covering nucleotides 1025–1044); antisense primer b-actin-2, 5*-CGC GAA TTC GTG AAC TTT GGG GGA TGC TC-3* (covering nucleotides 1396–1416); antisense primer b-actin-3, 5*-CGC AAG CTT CTG CTT GCT GAT CCA CAT CTG-3* (covering nucleotides 1147–1168); and sense primer b-actin-4, 5*-CGC AAG CTT AGT TGC GTT ACA CCC TTT CTT G-3* (covering nucleotides 1229–1250); and two primers derived from the cDNA sequences of the proa1 chain of type III procollagen: sense primer COL3A1-1, 5*-CGG AAT TCT TGG GAT TGC TGG GAT CAC T-3* (covering nucleotides 2934–2954) and antisense primer COL3A1-2, 5*-CGC GAA TTC GTG AAC TTT GGG GGA TGC TC-3* (covering nucleotides 3844–3866). The primers numbered 1 and 2 in each case contained a site for the EcoRI restriction endonuclease, and primers numbered 3 and 4 a site for HindIII. The oligonucleotide primers were synthesized in an Applied Biosystems DNA synthesizer (Department of Biochemistry, University of Oulu). Two PCRs were first carried out for the lysyl oxidase and bactin cDNAs, one with primers 1 and 3 and the other with primers 2 and 4. PCR was performed at a final concentration of 11 PCR buffer (10 mM Tris–HCl, pH 8.8, 1.5 mM MgCl2 , 50 mM KCl, 0.1% Triton X-100) (Dynazyme), 200 mM dNTPs, 10 pmol of each primer, 1 unit of thermostable DNA polymerase (Dynazyme) in a total volume of 50 ml. The amplification profile involved denaturation at 947C for 1 min 30 s, primer annealing at 54–647C for 1 min, and extension at 727C for 1 min. The amplified products were digested with HindIII (Pharmacia) and then ligated at the HindIII sites. The ligated fragment was then used as a template in a PCR with primers 1 and 2. The product now contained a deletion. In the amplification reaction for the type III procollagen cDNA only primers 1 and 2 were used and the deletion was generated by digesting the PCR product with TaqI restriction enzyme (Pharmacia), thereby removing a small part from the center of the PCR product. The PCR products with deletions were subcloned into plasmid Bluescript SK (Stratagene) as described (25). The plasmids were purified by the CsCl method (25). These plasmids were used as templates for transcription by the T3 or T7 polymerase according to the transcription protocol of Ambion, Inc. The resulting control RNA was quantified by two methods: absorbance at 260 nm and addition of 5 mCi of [32P]UTP (400 Ci/mmol, Amersham) to the in vitro transcription reaction and determination of its incorporation by trichloroacetic acid precipitation as advised by Ambion, Inc. Total cellular RNA was isolated from the cells by the method of acid guanidinium thiocyanate–phenol–chloroform (28). cDNA synthesis and PCR amplification. A 30-ml reverse transcription reaction was carried out on 0.5–6 mg of total cellular RNA and an in vitro-synthesized control containing 7.5 1 108 molecules of RNA for type III procollagen, 11.1 1 108 molecules for lysyl oxidase, or 5.5 1 109 molecules for b-actin. The reaction mixture contained 11 first-strand buffer (50 mM Tris–HCl, pH 8.3, 5 mM KCl, 3 mM MgCl2) (Gibco BRL), 10 mM dithiothreitol, 0.5 mM dNTP, 4 pmol random primers (Gibco BRL), and 100 units of Moloney murine leu-
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LYSYL OXIDASE mRNA IN HUMAN MENKES AND OCCIPITAL HORN SYNDROME CELLS TABLE I
Lysyl Oxidase Activity and Number of mRNA Molecules per Picrogram of Total RNA for Lysyl Oxidase mRNA Produced by the Various Cell Lines
Cell line Control cells Embryonal skin fibroblasts HES Adult skin fibroblasts 9011 9505 20009 Group mean Menkes cells GMO 1057 GMO 220 GMO 3700 Group mean Occipital horn cells ED58 ED 3999 Group mean
Lysyl oxidase activity (dpm/106 cells)
2130
Number of lysyl oxidase mRNA molecules/ pg total RNAa
440 { 25b (4)c
1780 1100 1430 1640 { 420
430 360 340 390
{ { { {
120 90 50 90
(13) (6) (13) (36)
790 590 670 680 { 80
290 230 150 230
{ { { {
160 40 20 110
(11) (10) (8) (29)
630 400 520 { 100
290 { 110 (8) 190 { 30 (6) 240 { 100 (14)
a
The values of the control cells are the same as in Ref. 27. SD. c Number of values used in calculating the mean. b
kemia virus reverse transcriptase (Gibco BRL), and was incubated at 377C for 60 min. PCR was performed in a final concentration of 11 PCR buffer, 160 mM dNTPs, 10 pmol of each 3* primer, 10 pmol of each 32P end-labeled 5* primer, and 1 unit of thermostable DNA polymerase in a total volume of 50 ml. The amplification involved denaturation at 947C for 1 min 30 s, annealing at 607C for 1 min 20 s, and extension at 727C for 1 min 30 s. The oligonucleotides were labeled with T4 polynucleotide kinase and [g32P]ATP (3000 Ci/mmol, Amersham Corp.). The sizes of the PCR products were 568 and 508 bp for lysyl oxidase, 392 and 337 bp for b-actin, and 919 and 748 bp for type III procollagen. Quantitative analysis. A 10-ml aliquot of each PCR reaction mixture was electrophoresed on 6% (v/v) polyacrylamide gels. The gels were dried and autoradiographed on X-ray films (Kodak). Appropriate bands were cut out from the gel and radioactivity was determined with a liquid scintillation counter. The efficiency of amplification was determined using the formula log Y Å log A / n log(1 / R), where Y is the extent of amplification, A is the amount of initial material, R is the efficiency, and n is the number of cycles. It is possible to estimate Y with reasonable accuracy by measuring the extent of incorporation of 32P-labeled primer into the PCR product, and R can be deduced from the slope of the semilog plot (29, 30). The Statistica software package (StatSoft), specifically the Spjøtvoll and Stoline extension of the t test, was used for comparisons between cell lines and groups of cell lines. The test is a powerful, i.e., conservative, test that can be used for simultaneous comparison of multiple groups of unequal n (31). Lysyl oxidase activity assay. L-[4,6-3H]lysine-labeled purified chick embryo elastin substrate was prepared (32), and the lysyl oxidase activity of the cell culture media was measured with 125,000 cpm of elastin substrate in a total volume of 0.8 ml (32). The samples were incubated for 10 h at 377C, and the tritiated water formed
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during the incubation was isolated by vacuum distillation and quantified with a liquid scintillation counter (32).
RESULTS
Lysyl Oxidase Activity in the Medium of Cultured Fibroblasts In agreement with previous data (10, 12–15), a decreased level of lysyl oxidase activity was seen in the culture medium of all three Menkes cell lines and the two occipital horn cell lines (Table I). The enzyme activity in the Menkes cell lines ranged from 35 to 49% of the control mean and that of the two occipital horn cell lines from 23 to 37%. The mean enzyme activity in both groups was significantly below the control mean (P Å 0.006). Northern Analysis Human lysyl oxidase mRNA is found by Northern analysis in multiple forms with approximate sizes of about 5.5, 4.3 (major mRNA), 2.5, and 2.0 kb, although the 2.5- and 2.0-kb mRNAs are usually not resolved in short runs (22, 33). No qualitative differences were found in this pattern of mRNAs in the Menkes and occipital horn fibroblasts, but the quantity of all the mRNA species appeared to be decreased relative to the control cell lines (Fig. 1). When the same blot was hybridized to a probe for glyceraldehyde-3-phosphate dehydrogenase, no significant differences were seen between the cell lines (not shown). Quantitative Analysis and Internal Standard To obtain a more accurate and reliable measurement of the lysyl oxidase mRNA levels, a quantitative PCR method was used in which control RNAs for lysyl oxidase, b-actin, and type III procollagen, each containing a small deletion compared with the full-length cDNA
FIG. 1. Northern blot using lysyl oxidase cDNA probe. Total RNA from the cultured control skin fibroblasts (9011 and HES), fibroblasts of one occipital horn syndrome patient (ED58), and fibroblasts of two Menkes syndrome patients (GMO 3700 and GMO 220) was isolated, electrophoresed on a 1% agarose gel, and transferred onto membrane that was probed with a 32P-labeled lysyl oxidase cDNA probe, as described under Materials and Methods. The two main lysyl oxidase mRNA species of about 5.5 and 4.3 kb are shown.
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FIG. 2. Autoradiogram of the cRNA and target mRNA products for type III procollagen, lysyl oxidase, and b-actin. The cRNA and target mRNA products for type III procollagen, lysyl oxidase, and b-actin were separated by 6% polyacrylamide gel electrophoresis. Serial 1:2 dilutions of the cDNA mixture were amplified using specific oligonucleotides. One of the primers was 32P-end-labeled.
clones, were synthesized from plasmids. The deletion was made by PCR in the case of lysyl oxidase and bactin cDNA or by enzymatic digestion in the case of type III procollagen cDNA. First-strand cDNA was synthesized in the same tube for a mixture of control RNA and RNA isolated from the cell line. Dilution series of the cDNA mixtures were then coamplified in the same tube, and the reactions were terminated in the exponential phase of amplification. These control RNAs served as internal standards for the reverse transcription reactions and PCRs and were used to generate standard curves for quantifying specific target mRNAs from experimental samples. The PCR primers used were the same for the control RNAs and target mRNAs, and thus there were no primer efficiency differences between these templates. In fact, determination of the amplification efficiencies failed to distinguish between the control construct and the cDNA from the cellular RNA for all three mRNAs studied. The size differences between the PCR products permitted easy separation of the cRNA product from the target mRNA product by gel electrophoresis (Fig. 2). The amounts of radioactivity recovered from the excised gel band were plotted against the template concentrations. The reaction rates of the control RNA and the target mRNA amplifications were identical in the exponential phase of the PCR reaction. This allowed construction of a standard curve for the control RNAs and extrapolation to a copy number for the target mRNAs isolated from cultured cells. Quantification of the Lysyl Oxidase, b-Actin, and Type III Procollagen mRNAs in the Various Cell Types The numbers of lysyl oxidase mRNA molecules in the four control cell lines were very similar, ranging from
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about 340 molecules/pg total RNA in cell line 20009 to 440 molecules/pg in the HES cells (Table I). The mean value for the three Menkes cell lines studied was significantly lower (P Å 0.0001), about 60% of the control mean with the values ranging from about 150 molecules/pg RNA in the GMO 3700 cells to 290 molecules/ pg in the GMO 1057 cells (Table I). The mean for the two occipital horn cell lines was likewise about 60% of the control mean (P Å 0.001), the values being 190 and 290 molecules/pg. Thus the values in all three Menkes cell lines and two occipital horn cell lines were below that in the lowest control. The number of b-actin mRNA molecules per picogram of total RNA showed a larger variation among the four control lines (Table II). A slightly higher mean was found in the group of the Menkes cells, whereas the mean for the two occipital horn cell lines was decreased (P Å 0.0001). The lowest value among all the cell lines, about 2000 molecules/pg RNA, was nevertheless found in the control cell line 9011 (Table II). The number of mRNA molecules encoding type III procollagen (Table II) was very high, about 230% of the mean of the controls, in two of the three Menkes cell lines (GMO 220, P Å 0.0002; and GMO 3700, P Å 0.0094), whereas this number was not increased in the third line (GMO 1057, P Å 0.816). No increase was found in this value in the only occipital horn cell line
TABLE II
Number of mRNA Molecules per Picogram of Total RNA for b-Actin and Type III Procollagen in the Various Cell Lines
Cell line Control cells Embryonal skin fibroblasts HES Adult skin fibroblasts 9011 9505 20009 Group mean Menkes cells GMO 1057 GMO 220 GMO 3700 Group mean Occipital horn cells ED 58 ED 3999 Group mean a
Number of type Number of b-actin III procollagen mRNA molecules/ mRNA molecules/ pg total RNA pg total RNAa
5170 { 114b (7)c
380 { 20 (6)
2000 6600 5500 4500
{ { { {
400 1690 1600 2230
(9) (7) (3) (26)
470 670 980 590
120 120 500 310
(11) (7) (6) (30)
5250 4930 4230 4780
{ { { {
1530 430 1090 1110
(6) 460 { 50 (7) 1380 { 220 (7) 1350 { 160 (20) 1050 { 480
(6) (8) (3) (17)
2400 { 400 (9) 3400 { 530 (5) 2820 { 630 (13)
{ { { {
390 { 70 (7) 390 { 70 (7)
The values of the control cells are the same as in Ref. 27. SD. c Number of values used in calculating the mean. b
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LYSYL OXIDASE mRNA IN HUMAN MENKES AND OCCIPITAL HORN SYNDROME CELLS
analyzed, where the number was about 65% of the control mean and only slightly above that in the lowest control (Table II). DISCUSSION
The present data indicate that the numbers of lysyl oxidase mRNA molecules per picogram RNA were significantly decreased in the three cultured skin fibroblast lines from patients with the Menkes syndrome and the two from patients with the occipital horn syndrome. This effect was specific in the Menkes cell lines, in that no decrease was found in the number of b-actin mRNA molecules but rather a slight increase, whereas the cells from patients with the occipital horn syndrome also showed a decrease in the number of the bactin mRNA molecules. The data thus indicate that the decreases seen in the amounts of lysyl oxidase activity in these two diseases are not simply due to abnormal utilization of copper as a cofactor for the enzyme but are at least in part due to a pretranslational mechanism, either diminished transcription of the lysyl oxidase gene or impaired processing or stability of the mRNA. The decrease in the number of the mRNA molecules was not as marked as that in the enzyme activity, however, suggesting that additional mechanisms contribute to the low enzyme activity. One such mechanism might well be a poorer stability of the copperdeficient lysyl oxidase protein, as earlier studies have indicated a large decrease in the amount of this enzyme in cells from patients with the Menkes syndrome (13, 16) and occipital horn syndrome (13), and from the tortoiseshell mouse (16), a murine analogue of the Menkes syndrome (2). The present data suggesting a diminished number of lysyl oxidase mRNA molecules agree with figures recently reported in cells from one Menkes patient and one tortoiseshell mouse (16), except that the magnitudes of the decreases obtained on that occasion, using only Northern blotting, were much larger than those determined here. The data disagree with a recent report by another group (24), however, who found no decrease in the amount of the lysyl oxidase mRNA in cells from three Menkes patients by Northern blotting. Because the number of lysyl oxidase mRNA molecules in one of our Menkes cell lines was only about 15% below that in the lowest control, it is evident that the decreases in cells from some patients cannot be detected by Northern blotting. Furthermore, it seems likely that some Menkes cell lines may have lysyl oxidase mRNA molecule numbers that are within the control range. It is now well established that the basic defect in the Menkes syndrome and the occipital horn syndrome is a mutation in an X-chromosomal gene encoding a coppertransporting ATPase (4–7). The present and previous
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data (16) indicate that the consequences of this defect extend far beyond those which could be expected on the basis of the simple alteration in the availability of copper to the enzymes that utilize this cation, such as lysyl oxidase. A previous study has reported an increased level of mRNA for the proa1 chain of type I procollagen in cells from a Menkes patient and a tortoiseshell mouse (16). The present data extend this finding by indicating that the number of mRNA molecules for type III procollagen was increased in two of the three Menkes cell lines studied. Accordingly, the ratio of lysyl oxidase mRNA molecules to type III procollagen mRNA molecules in these two cell lines was only about 25% (GMO 220) or 17% (GMO 3700) of that in the controls. The amount of elastin mRNA has also been reported to be markedly altered in cells from a Menkes patient and a tortoiseshell mouse (16), but the change is opposite in direction to those seen in the amounts of type I procollagen (16) and type III procollagen mRNAs, in that the amount of elastin mRNA was markedly decreased. An additional abnormality found in the present study was that the number of b-actin mRNA molecules was decreased in cells from patients with the occipital horn syndrome. The mechanisms that lead to these changes in the various mRNA levels in the Menkes and occipital horn syndrome cells are currently unknown. Analysis of additional Menkes cell lines will allow the use of statistical tests to determine whether cell lines from patients diagnosed with the Menkes syndrome can be classified into two or more categories. Should multiple categories exist, it is possible that variance in the levels of expression of some of the mRNAs for extracellular matrix proteins could explain some or all of the phenotypic variance. ACKNOWLEDGMENTS We are grateful to Aira Harju and Riitta Poloja¨rvi for their technical assistance. The work was supported by grants from the Medical Research Council of the Academy of Finland, the Sigrid Juse´lius Foundation and the Paulo Foundation.
REFERENCES 1. Kivirikko, K. I., and Kuivaniemi, H. (1987) in Connective Tissue Disease (Uitto, J., and Perejda, A. J., Eds.), pp. 263–292, Dekker, New York. 2. Danks, D. M. (1993) in Connective Tissue and Its Heritable Disorders: Molecular, Genetic and Medical Aspects (Royce, P. M., and Steinmann, B., Eds.), pp. 487–506, Wiley–Liss, New York. 3. Kivirikko, K. I. (1993) Ann. Med. 25, 113–126. 4. Vulpe, C., Levinson, B., Whitney, S., Packman, S., and Gitschier, J. (1993) Nat. Genet. 3, 7–13. 5. Chelly, J., Tumer, Z., Tønnesen, T., Petterson, A., IshikawaBrush, Y., Tommerup, N., Horn, N., and Monaco, A. P. (1993) Nat. Genet. 3, 14–19. 6. Mercer, J. R. B., Livingston, J., Hall, B., Paynter, J. A., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemienak, D., and Gover, T. W. (1993) Nat. Genet. 3, 20–25.
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21. 22. 23. 24. 25.
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