CaMKII inhibition in human primary and pluripotent stem cell-derived chondrocytes modulates effects of TGFß and BMP through SMAD signaling.

OBJECTIVE: Upregulation of calcium/calmodulin-dependent kinase II (CaMKII) is implicated in the pathogenesis of osteoarthritis (OA) and reactivation of articular cartilage hypertrophy. However, direct inhibition of CaMKII unexpectedly augmented symptoms of OA in animal models. The role of CaMKII in OA remains unclear and requires further investigation. METHODS: Analysis of CaMKII expression was performed in normal human and OA articular chondrocytes, and signaling mechanisms were assessed in articular, fetal and Pluripotent Stem Cell (PSC)-derived human chondrocytes using pharmacological (KN93), peptide (AC3-I) and small interfering RNA (siRNA) inhibitors of CaMKII. RESULTS: Expression levels of phospho-CaMKII (pCaMKII) were significantly and consistently increased in human OA specimens. BMP2/4 activated expression of pCaMKII as well as COLII and COLX in human adult articular chondrocytes, and also increased the levels and nuclear localization of SMADs1/5/8, while TGFß1 showed minimal or no activation of the chondrogenic program in adult chondrocytes. Targeted blockade of CaMKII with specific siRNAs decreased levels of pSMADs, COLII, COLX and proteoglycans in normal and OA adult articular chondrocytes in the presence of both BMP4 and TGFß1. Both human fetal and PSC-derived chondrocytes also demonstrated a decrease of chondrogenic differentiation in the presence of small molecule and peptide inhibitors of CaMKII. Furthermore, immunoprecipitation for SMADs1/5/8 or 2/3 followed by western blotting for pCaMKII showed direct interaction between SMADs and pCaMKII in primary chondrocytes. CONCLUSION: Current study demonstrates a direct role for CaMKII in TGF-ß and BMP-mediated responses in primary and PSC-derived chondrocytes. These findings have direct implications for tissue engineering of cartilage tissue from stem cells and therapeutic management of OA.

Inhibition of collagen gene expression in systemic sclerosis dermal fibroblasts by mithramycin.

BACKGROUND: The anti-tumour antibiotic mithramycin is also a potent inhibitor of fibrosis after glaucoma surgery. This drug displays high affinity binding to GC-rich sequences in DNA, including those present in the promoter of the gene encoding the alpha1 chain of type I collagen (COL1A1). OBJECTIVE: To evaluate the effects of mithramycin on COL1A1 expression in systemic sclerosis fibroblasts. METHODS: Confluent cultures of dermal fibroblasts from patients with recent onset diffuse systemic sclerosis were treated with mithramycin in vitro. Cell viability and protein expression were examined by fluorescence and confocal imaging. Type I collagen production was analysed by confocal imaging and metabolic labelling. COL1A1 messenger RNA levels and stability were assessed by northern hybridisation, and COL1A1 transcription was examined by transient transfections. RESULTS: Treatment of systemic sclerosis fibroblasts with mithramycin (10-100 nmol/l) did not cause significant cytotoxicity. Type I collagen biosynthesis decreased by 33-40% and 50-70% in cells cultured with mithramycin at 10 nmol/l and 100 nmol/l, respectively. Mithramycin at 50 nmol/l decreased COL1A1 mRNA levels by 40-60%. The effects of mithramycin on collagen gene expression were mediated by transcriptional and post-transcriptional mechanisms as shown by the reduction of COL1A1 promoter activity and by a decrease in the stability of these transcripts, respectively. CONCLUSIONS: Mithramycin causes potent inhibition of collagen production and gene expression in systemic sclerosis dermal fibroblasts in vitro in the absence of cytotoxic effects. These results suggest that this drug may be an effective treatment for the fibrotic process which is the hallmark of systemic sclerosis.

Role of protein kinase C-delta in the regulation of collagen gene expression in scleroderma fibroblasts.

Working with cultured dermal fibroblasts derived from control individuals and patients with systemic sclerosis (SSc), we have examined the effects of protein kinase C-delta (PKC-delta) on type I collagen biosynthesis and steady-state levels of COL1A1 and COL3A1 mRNAs. Rottlerin, a specific inhibitor of PKC-delta, exerted a powerful, dose-dependent inhibition of type I and type III collagen gene expression in normal and SSc cells. Optimal rottlerin concentrations caused a 70-90% inhibition of type I collagen production, a >80% reduction in COL1A1 mRNA, and a >70% reduction in COL3A1 mRNA in both cell types. In vitro nuclear transcription assays and transient transfections with COL1A1 promoter deletion constructs demonstrated that rottlerin profoundly reduced COL1A1 transcription and that this effect required a 129-bp promoter region encompassing nucleotides -804 to -675. This COL1A1 segment imparted rottlerin sensitivity to a heterologous promoter. Cotransfections of COL1A1 promoter constructs with a dominant-negative PKC-delta expression plasmid showed that suppression of this kinase silenced COL1A1 promoter activity. The results indicate that PKC-delta participates in the upregulation of collagen gene transcription in SSc and suggest that treatment with PKC-delta inhibitors could suppress fibrosis in this disease.

Physical mapping of mouse collagen genes on chromosome 10 by high-resolution FISH.

Fluorescence in situ hybridization (FISH) on mechanically stretched chromosomes (MSCs) and extended DNA fibers enables construction of high-resolution physical maps by accurate ordering and orienting genomic clones as well as by measuring physical lengths of gaps and overlaps between them. These high-resolution FISH targets have hitherto been used mainly in the study of the human genome. Here we have applied both MSCs and extended DNA fibers to the physical mapping of the mouse genome. At first, five mouse collagen genes were localized by metaphase-FISH: Col10a1 to chromosomal bands 10B1-B3; Col13a1 to 10B4; and Col6a1, Col6a2, and Col18a1 to 10B5-C1. The mutual order of the genes, centromere--Col10a1--Col13a1--Col6a2--Col6a1--Col18a1--telomere, was determined by FISH on metaphase chromosomes, MSCs, and extended DNA fibers. To our knowledge, this is the first time mouse metaphase chromosomes have been stretched and used as targets for FISH. We also used MSCs to determine the transcriptional orientations, telomere--5'-->3'--centromere, of both Col13a1 and Col18a1. With fiber-FISH, Col18a1, Col6a1, and Col6a2 were shown to be in a head-to-tail configuration with respective intergenic distances of about 350 kb and 90 kb. Comparison of our physical mapping results with the homologous human data reveals both similarities and differences concerning the chromosomal distribution, order, transcriptional orientations, and intergenic distances of the collagen genes studied.

CCAAT binding transcription factor binds and regulates human COL1A1 promoter activity in human dermal fibroblasts: demonstration of increased binding in systemic sclerosis fibroblasts.

OBJECTIVE: To determine the binding factors that interact with the proximal promoter region of the human type I collagen gene, COL1A1, and to examine their involvement in its transcriptional regulation in normal and systemic sclerosis (SSc) dermal fibroblasts. METHODS: Nuclear extracts from dermal fibroblasts from 4 patients with SSc and 4 age- and sex-matched control individuals were examined by electrophoresis mobility shift assays with a COL1A1 promoter fragment encompassing nucleotides -174 to -50 bp. Supershift assays with antibodies specific to various transcription factors, and competition experiments using consensus, wild-type, or mutated oligonucleotides corresponding to their specific binding sites, were performed. The effects of specific oligonucleotides as "intracellular competitors" were examined by transient transfection experiments in SSc fibroblasts using a COL1A1 construct containing -174 bp of the promoter. RESULTS: The findings demonstrate that the CCAAT binding transcription factor (CBF) binds the proximal CCAAT box located at -100 to -96 bp, but not the distal CCAAT box at -125 to -121 bp, of the human COL1A1 promoter in both SSc and normal fibroblasts. CBF binding activity was 3-5-fold higher in the SSc fibroblasts. Moreover, the promoter activity of the -174-bp COL1A1 construct was decreased by up to 50% when specific oligonucleotides were used as "intracellular competitors." In addition, Sp1 and Sp3 were other transcription factors found to be involved in the formation of the DNA-protein complexes within this region of the COL1A1 promoter. CONCLUSION: These results indicate that the transcription factor CBF binds the human COL1A1 proximal promoter region in human dermal fibroblasts, and its binding activity is higher in SSc fibroblasts.

Inhibition of type I collagen gene expression in normal and systemic sclerosis fibroblasts by a specific inhibitor of geranylgeranyl transferase I.

OBJECTIVE: To examine the effects of specific inhibition of geranylgeranyl transferase I on the expression of types I and III collagen genes in normal and systemic sclerosis (SSc) dermal fibroblasts in vitro. METHODS: Fibroblasts from 2 normal subjects and 4 SSc patients were incubated with 2-10 microM of GGTI-298, a specific geranylgeranyl transferase inhibitor. Type I collagen and fibronectin production were determined by enzyme-linked immunosorbent assay. Steady-state messenger RNA (mRNA) levels for alpha1(I), alpha2(I), and alpha1(III) collagens and fibronectin were assessed by Northern hybridization, and the transcription of the alpha1(I) collagen gene was examined by transient transfections with a reporter construct containing -5.3 kb of the gene. RESULTS: GGTI-298 caused a dose-dependent inhibition of type I collagen production and a reduction in the steady-state levels of alpha1(I), alpha2(I), and alpha1(III) mRNA in normal and SSc cells. A 60-70% inhibition of type I collagen production and a 70-80% reduction in the mRNA levels for alpha1(I), alpha2(I), and alpha1(III) were observed at 10 microM GGTI-298. In contrast, the expression of fibronectin, cyclooxygenase 1, and GAPDH was not affected. The effects on alpha1(I) collagen mRNA resulted from a profound reduction in transcription of the alpha1(I) collagen gene promoter. GGTI-298 did not affect cellular viability or morphology. CONCLUSION: These results demonstrate that specific inhibition of geranylgeranyl prenylation causes a potent and selective inhibition of expression of the genes encoding types I and III collagens, without affecting cellular viability. The findings indicate that inhibition of geranylgeranyl prenylation should be further studied as a potential therapeutic approach for SSc and other fibrosing diseases.

A novel de novo mutation in the triple helix of the COL6A3 gene in a two-generation Italian family affected by Bethlem myopathy. A diagnostic approach in the mutations' screening of type VI collagen.

Bethlem myopathy is an autosomal dominant inherited disease producing a mild neuromuscular disorder, characterized mainly by muscular weakness and multiple joint contractures. Bethlem myopathy is caused by mutations in one of the three chains of collagen type VI. Here we report the clinical description and the molecular characterization of the defect in a two-generation Italian family in which a Gly-->Arg substitution disrupts the triple helix structure of the alpha 3 chain of collagen type VI, an ubiquitous glycoprotein of the extracellular matrix. In this family the identification of the mutation also allowed one to exclude the disease in the grandfather. It is noteworthy that the father of the proband carries a de novo mutation, the first described for Bethlem myopathy.

A heterozygous splice site mutation in COL6A1 leading to an in-frame deletion of the alpha1(VI) collagen chain in an italian family affected by bethlem myopathy.

Bethlem myopathy is a mild neuromuscular disorder with proximal muscular weakness and early flexion contractures. It is an autosomal dominant disease due to mutations in type VI collagen genes. We found a T-->C substitution at the +2 position of COL6A1 intron 14 in a family, leading to skipping of exon 14 and an in-frame deletion of 18 amino acids in the triple-helical domain of the alpha1(VI) collagen chain. The deletion included a cysteine residue believed to be involved in the assembly of type VI collagen dimers intracellularly, prior to the protein secretion. Analysis of the affected fibroblasts showed that the shortened alpha1(VI) collagen chains were synthesized but not secreted by the cells and that the amount of type VI collagen microfibrils deposited by the cells was reduced. The results suggest that the clinical phenotype is due to a reduction in the level of type VI collagen in the extracellular matrix.

B-MYB transactivates its own promoter through SP1-binding sites.

B-MYB is an ubiquitous protein required for mammalian cell growth. In this report we show that B-MYB transactivates its own promoter through a 120 bp segment proximal to the transcription start site. The B-MYB-responsive element does not contain myb-binding sites and gel-shift analysis shows that SP1, but not B-MYB, protein contained in SAOS2 cell extracts binds to the 120 bp B-myb promoter fragment. B-MYB-dependent transactivation is cooperatively increased in the presence of SP1, but not SP3 overexpression. When the SP1 elements of the B-myb promoter are transferred in front of a heterologous promoter, an increased response to B-MYB results. In contrast, c-MYB, the prototype member of the Myb family, is not able to activate the luciferase construct containing the SP1 elements. With the use of an SP1-GAL4 fusion protein, we have determined that the cooperative activation occurs through the domain A of SP1. These observations suggest that B-MYB functions as a coactivator of SP1, and that diverse combinations of myb and SP1 sites may dictate the responsiveness of myb-target genes to the various members of the myb family.

Alterations in the regulation of expression of the alpha 1(I) collagen gene (COL1A1) in systemic sclerosis (scleroderma).

At present, the mechanisms that regulate the expression of collagen genes in normal and pathologic fibroblasts are not known. Thus, the detailed study of transcriptional regulation of COL1A1 in SSc cells will increase our current understanding of the pathophysiology of fibrotic diseases. These studies will yield valuable information regarding the important biological process of regulation of collagen gene expression under normal and pathologic conditions, a process that has remained elusive despite intense recent investigations. It is now evident that persistent overproduction of collagen is responsible for the progressive nature of tissue fibrosis in SSc. Up-regulation of collagen gene expression in SSc fibroblasts appears to be a critical event in this process. The coordinate transcriptional activation of numerous collagen genes suggests a fundamental alteration in the regulatory control of gene expression in SSc fibroblasts. Trans-acting nuclear factors which bind to cis-acting elements in enhancer (intronic) and promoter regions of the genes modulate the basal and inducible transcriptional activity of the collagen genes. The identification of the nuclear transcription factors that regulate normal collagen gene expression may provide promising approaches to the therapy of this incurable disease.

Identification of a polymorphic CA repeat in the COL6A2 gene on human chromosome 21q22.3.

We have identified a CA repeat in intron 10 of the human alpha 2(VI) collagen gene (COL6A2), located on chromosome 21q22.3. At least seven alleles ranging from 14 to 20 repeats have been demonstrated by the polymerase chain reaction from genomic DNA of 71 random unrelated healthy persons.

Presence of different collagens and collagen mRNAs during embryogenesis and in adult tissues of the sea urchin Paracentrotus lividus.

The presence of four different collagen genes had been previously described in the sea urchin genome and four different cDNAs had been cloned and sequenced. Two of them code for 140 and 300 KDaltons proteins, belonging to the fibrillar collagens, and the other two families code for two type IV collagens with a molecular weight of about 210 KDaltons. In this paper immunological evidence is provided for the presence in the developing P. lividus sea urchin embryo of at least seven major collagen proteins. Western blot analyses, carried out by means of specific polyclonal antibodies, show a series of collagenase sensitive bands, with molecular weights ranging from 55 to 200 KDaltons, which are present from eggs to plutei. Northern blot analyses show the presence of the previously described 6 and 9 Kb RNA bands from oocytes till plutei; in the later stages two other collagen RNAs are detected. The presence of two sets of genes coding for the 6 Kb mRNAs, differentially expressed during development, is also discussed. Immunofluorescence histological analyses show the location of collagen in gonads, oocytes, eggs, embryos and adult tissues.

Characterization of the human alpha 1(VI) collagen promoter and its comparison with human alpha 2(VI) promoters.

From a human cosmid library, we isolated a clone (5B) with an insert of 32 kb, encoding the amino-terminal and the 5'-end flanking region of the alpha 1(VI) collagen gene. Exon 1 was found to be 194 bp and contain the 5' untranslated region plus 97 bp coding sequence. Exon 2 consists of 130 bp, a size that is conserved across the chicken and mouse species. S1-nuclease-protection assays and primer-extension analysis, using mRNA from human dermal fibroblasts, show the presence of multiple transcription start sites located in a region of approximately 20 nucleotides. Canonical TATA and CAAT boxes, as found in the chicken and mouse alpha 1 promoters, were absent in the human alpha 1(VI) promoter. The promoter region from positions -1 to -190, is a polypyrimidine/polypurine-rich region containing 12 CCCTCCCC (CT element consensus) sequences and has multiple potential binding sites for the Sp1, and AP2 transcription factors. These regulatory proteins bind to the alpha 2(VI) promoters [Saitta, B. & Chu, M.-L. (1994) Eur. J. Biochem. 223, 675-682]. To test the transcriptional activity of the alpha 1 promoter, transient transfection experiments of the DNA constructs were performed in human dermal fibroblasts and in human fibrosarcoma (HT1080) cell lines. The DNA constructs drive the expression of the chloramphenicol acetyl transferase (CAT) gene. The results show strong CAT activity for the constructs at positions -1700, -298 and -257, while low activity was found for the constructs at positions -4400, -142 and -5 when transfected in fibroblasts. The experiments also identified positive and negative regulatory regions in the alpha 1(VI) promoter CAT constructs when transfected in fibroblasts, but did not identify them in the fibrosarcoma cells.

Head to tail organization of the human COL6A1 and COL6A2 genes by fiber-FISH.

Two type VI collagen genes, COL6A1 and COL6A2, both map to 21q22.3, but the order, distance, and organization of these two genes relative to one another were not known. Recently developed high-resolution fluorescence in situ hybridization (FISH) techniques have great potential to facilitate the construction of fine-resolution maps of telomeric regions where gene density is high. Here we have determined the distance separating the COL6A1 and COL6A2 genes (150 kb), the size of the COL6A1 gene (29 kb); and the 5'-3' orientation of these genes (5' COL6A1 3'-5' COL6A2 3') using fiber-FISH.

Myocardial technetium-99m sestamibi single-photon emission tomography as a prognostic tool in coronary artery disease: multivariate analysis in a long-term prospective study.

To date several studies have evaluated the accuracy of thallium-201 myocardial scan in risk stratification of coronary artery disease (CAD), while reports using technetium-99m methoxyisobutylisonitrile (MIBI), a tracer particularly suited to single-photon emission tomographic (SPET) imaging, are lacking. To rectify this omission, a prospective study was started in 1988 and at present 176 consecutive, and thus unselected, patients have been enrolled. All of them have been submitted to stress-rest MIBI SPET for the diagnosis or evaluation of CAD; 147 patients (121 males and 26 females, aged 53 +/- 9 years) have completed a surveillance period of at least 36 months following the scintigraphic study (range 36-60 months, mean 43). Sixty-one patients had a documented previous myocardial infarction. The mean pre-test likelihood of CAD was 44% in the patients without prior infarction. The main anamnestic, clinical, EKG and scintigraphic findings were evaluated and statistically correlated with the incidence of ensuing cardiac events using both univariate (chi-square test) and multivariate analysis (logistic regression model). Twenty-nine patients suffered from a cardiac event during the follow-up period (i.e. three cardiac deaths, six myocardial infarctions and 20 cases of unstable angina). Statistical multivariate analysis identified MIBI scan as the only highly significant and independent prognostic predictor [P = 0.006, relative risk (RR) = 17.62]. In detail, the most important scintigraphic parameters were the presence of a reversible defect (P = 0.0089, RR = 5.11) and the extension of the stress perfusion defect (P = 0.0255, RR = 3.27). The presence of typical angina proved to be a slightly significant predictor (P = 0.051, RR = 2.45), while no other examined parameter showed a significant correlation with a bad prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Usefulness of 99mTc-MIBI stress myocardial SPECT bull's-eye quantification in coronary artery disease.

99mTc-methoxy-isobutyl isonitrile (MIBI) myocardial SPECT quantification performed using a Bull's-eye polar map, was evaluated and compared with visual analysis in 120 patients with proven or suspected CAD. The study series comprised 106 men and 14 women, age 37-75 years (mean 51 +/- 6), 68 of whom had had a prior myocardial infarction. Coronary angiography was taken as the gold standard: one-vessel disease was present in 24 patients, two-vessel disease in 39, and three-vessel disease in 44, whereas no significant stenosis was documented in 13 cases. Forty age-matched subjects (26 men, 14 women), with less than a 5% chance of having CAD, were enrolled to establish the normal database for males and females. ROC analysis was used to calculate the optimal thresholds for the definition of the disease extension in each vascular territory of the Bull's-eye polar map: 10% for LAD, 8% for LCX, and 20% for RCA territory. The sensitivity/specificity ratio of the scintigraphy was: 75/82% with the visual and 78/74% with the quantitative analysis for LAD; 60/90% with visual and 72/64% with visual and 70/62% with quantitative analysis for RCA territory. The sensitivity/specificity ratios for the CAD diagnosis were similar with the visual and the Bull's-eye analysis in 92/61% and 93/61% respectively. Bull's-eye analysis agreed with visual analysis in 296/360 vessels. Two and three-vessel disease were most frequently observed using the Bull's-eye approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of exercise-induced alterations of cardiac physiology on nuclear perfusion imaging.

The effects produced on nuclear perfusion images by exercise-induced changes in the heart and respiration rate and possible transient myocardial stunning are not well understood. In this study we attempted to indirectly evaluate the potential artifacts created by exercise-induced changes in cardiac physiology. Twenty patients with prior myocardial infarction and suspected peri-infarct ischemia were studied by 99mTc-MIBI imaging. Two SPECT perfusion studies were performed after 99mTc-MIBI administration at rest. The first acquisition was carried out 90 minutes after injection of the tracer. Immediately afterwards, the patients underwent a stress test followed by a second acquisition (exercise stress superimposed on rest tracer distribution). A standard stress perfusion scan was also performed 48 hours later. Superimposed exercise stress produced artifactual defects in the resting distribution of the tracer in 15 out of 20 patients (68 of 360 segments). Standard stress images demonstrated concordant defects in 48 of these segments, indicating the concomitant presence of ischemia and stunning. This study indicates that exercise-induced changes in cardiac physiology may result in artifactual perfusion defects in scintigraphic images acquired shortly after the stress.

Two promoters control the transcription of the human alpha 2(VI) collagen gene.

Our previous studies have demonstrated that the human alpha 2(VI) collagen gene produces four mRNA species with different 5'-untranslated regions [Saitta, B., Timpl, R. & Chu, M.-L. (1992) J. Biol. Chem. 267, 6188-6196]. The major mRNA species initiates from exon 1, located at the most 5' end, whereas three minor mRNAs start from an alternative exon, 1A, located 657 bp downstream of exon 1. In this study, we have investigated whether or not these different mRNAs are transcribed from two separate promoters. DNA fragments preceding exons 1 and 1A were fused with a reporter gene for chloramphenicol acetyl transferase (CAT) and transfected into human dermal fibroblasts and fibrosarcoma HT1080 cells. Strong CAT activity in both cell types was observed using a construct containing DNA from nucleotide -502 to + 115 preceding exon 1. The CAT activity of a construct containing nucleotide +514 to +894 preceding exon 1A was almost as high as that of the former construct, indicating the presence of two promoters, P1 and P2, preceding exons 1 and 1A, respectively. Transient transfection assays also identified positive and negative regulatory regions for the P1 promoter, located from nucleotide -2152 to -1384 and from nucleotide -1383 to -503, respectively. A negative regulatory region located at nucleotide +116 to +513 was found for the P2 promoter. This region strongly inhibits the P2 promoter in dermal fibroblasts, and thus may be responsible for the low expression of the endogenous exon-1A-containing mRNAs in these cells. Footprinting analysis of the two promoters with purified Sp1 protein and AP2 protein extract showed several sites of DNA-protein interaction. The specificity of these sites was confirmed by competition experiments using consensus Sp1 and AP2 oligonucleotides. The results thus demonstrate that the human alpha 2(VI) collagen gene contains two promoters, which are regulated by positive and negative cis-acting DNA elements and trans-acting factors.

Dopamine receptor SPET imaging in Parkinson's disease: a [123I]-IBZM and [99mTc]-HM-PAO study.

Single photon emission tomography (SPET) with the novel ligand [123I]-IBZM was used to image central dopamine D2 receptors in Parkinson's disease (PD) patients. The aim was to assess basal ganglia (BG) receptor densities in relation to the response to L-dopa therapy. To better characterize the clinical potential of [123I]-IBZM SPET, each patient underwent a second study with the regional perfusion tracer [99mTc]-HM-PAO. Tracer activity ratios were calculated for caudate and putamen with mean activity over the cerebellar hemispheres as internal standard. In PD patients we found a significant decline of mean caudate [123I]-IBZM activity, as compared with age-matched control subjects. However, when patients were grouped according to their therapeutic behavior, the [123I]-IBZM uptake in BG ganglia regions of the PD patient group with a poor and fluctuating response to L-dopa was significantly reduced from mean values of patients with a sustained response to L-dopa therapy. [99mTc]-HM-PAO caudate and putamen uptake indexes in PD were similar to control values, even in patients with deteriorated therapeutic response. Our results indicate that BG D2 receptor alterations in PD may contribute to the altered response to L-dopa.

Human alpha 2(VI) collagen gene. Heterogeneity at the 5'-untranslated region generated by an alternate exon.

Cosmid clones containing the 5' region of the human alpha 2(VI) collagen gene have been isolated and characterized. DNA sequencing indicates that the signal peptide and the amino-globular domain are encoded by four exons of 142, 596, 21, and 66 base pairs (bp). However, S1 nuclease and primer extension analyses show that the transcription start site is not present in the 142-bp exon. Two different 5' cDNA clones are generated by the anchored polymerase chain reaction. Using the 5' cDNA clones as probes, two untranslated exons (1, 1A) are found 12 kilobase pairs upstream of the first coding exon. These two exons are alternatively used in human fibroblasts, and most transcripts contain exon 1 sequence. Exon 1 shows, by primer extension and S1 nuclease protection assay, two major and several minor transcription start sites. The promoter region contains a canonical TATA box, seven GGGCGG sequences, two possible CAAT boxes, and two sequences resembling AP2 binding sites. Exon 1A contains three alternative splice donor sites and is located 650 bp downstream of exon 1. The most 3' splice donor site of exon 1A is found within an Alu repeat sequence. Exon 1A is preceded by five GGGCGG sequences and one resembling the AP2 binding site although neither TATA or CAAT boxes are found. Two additional GGGCGG sequences are located at the beginning of exon 1A. This study establishes that the human alpha 2(VI) collagen gene is 36 kilobase pairs long and contains 30 exons. The 5'-untranslated and promoter regions are significantly different from the corresponding segments of the chicken gene. The human gene produces by alternative processing multiple mRNAs differing in the 5'-untranslated region as well as the 3'-coding and noncoding sequences.