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.

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.

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.

Human nidogen gene: structural and functional characterization of the 5'-flanking region.

Nidogen is a sulfated multifunctional glycoprotein present in basement membranes. In this study, we have cloned the 5'-flanking region of the human nidogen gene. Initially, an approximately 35-kb DNA clone (NCos4) was isolated from a human cosmid genomic library. Southern hybridization of EcoRI-digested NCos4 allowed isolation of a 3.7-kb fragment, which was shown to contain a portion of intron 1, the entire exon 1, and approximately 0.9 kb of 5'-flanking sequences of the nidogen gene. Nucleotide sequencing of the 5'-flanking DNA revealed the presence of two canonic CCAAT consensus sequences in the antisense strand and a potential variant of the TATA motif, TATTT, in the sense strand. One putative AP-2 and six putative SP1 binding sites were also present. To test the functional promoter activity of the 5'-flanking genomic DNA, two nidogen promoter/CAT reporter gene constructs, with the promoter segment spanning from -864 to -1 and from -534 to -1, respectively, were developed and analyzed in transient transfections of human and mouse cell cultures. Both constructs showed clearly detectable promoter activity, and the activity of the larger construct could be up-regulated by 12-O-tetradecanoyl phorbol 13-acetate up to 2.5 times. The results indicate that the nidogen promoter/CAT gene constructs developed in this study provide a means to examine the transcriptional regulation of nidogen gene expression in human diseases of the basement membrane zone.

Regulation of elastin gene expression: evidence for functional promoter activity in the 5'-flanking region of the human gene.

Analysis of nucleotide sequences in the 5'-flanking region of the human elastin gene has revealed several unusual features, suggesting that regulation of elastin gene expression is complex. To identify any cis-acting regulatory promoter elements, a 35-kb fragment of DNA (CosE) was isolated from a human genomic cosmid library by hybridizations with a human elastin cDNA. Southern blots of EcoRI digests of CosE DNA, utilizing a 5'-end labeled 21-mer oligonucleotide corresponding to the signal sequence of elastin, revealed the presence of a single 7.8-kb genomic fragment. Partial dideoxynucleotide sequencing of this EcoRI genomic subclone revealed that it extended approximately 2.5 kb 3' of the translation initiation site (ATG), encompassing exon 1 and a portion of the first intron, while the remaining DNA encompassed the 5'-flanking region. Exonuclease III digestion (3'----5') was performed to remove sequences of the first intron and first exon, including the ATG site. One clone, approximately 5 kb in size, had the 3' end located 14 bp upstream of the ATG site. A 462-bp 3' portion of this 5-kb fragment was subcloned into a Bluescript/CAT chimeric plasmid (pBS0CAT) to generate an elastin gene promoter/CAT reporter gene construct (pEP6CAT). Transient transfection experiments with pEP6CAT using human skin fibroblasts, human HT-1080, mouse NIH-3T3, or freshly isolated neonatal rat aortic smooth muscle cells revealed significant CAT activity in each cell line. These results suggest that the 5'-flanking region of the elastin gene contains the cis-acting regulatory elements necessary for transcription. The chimeric plasmid pEP6CAT provides a means to study the transcriptional control of elastin gene expression by exogenous affector molecules, as well as in human dermatologic diseases.

[99mTc]-HM-PAO SPECT in Parkinson's disease.

Thirty-six patients affected by Parkinson's disease were studied using single photon emission computed tomography (SPECT) and [99mTc]-HM-PAO as a tracer. The scanning procedure was performed 16-24 h after discontinuation of specific therapy. Tracer activity ratios were determined in 10 pairs of cerebellar, cortical, and subcortical regions. Data were compared with those of 10 age-matched controls. Most of the regions examined did not show any relevant change between parkinsonian and control subjects. Notably, mean activity in striatal regions were similar in the two groups. Increased activity in caudate-putamen was found in patients who were on chronic DOPA therapy. Side-to-side asymmetries in the basal ganglia increased with the severity of the disease. Significant reductions of tracer uptake, from control values, were observed bilaterally in the parietal cortex. These deficits were more pronounced in patients with mental deterioration and in subjects who had been chronically treated with anticholinergic drugs. Parietal perfusion deficits in parkinsonian patients resemble those described in Alzheimer's dementia. These findings suggest that the heterogeneous alterations of regional cerebral blood flow (rCBF) in parkinsonian patients reflect the multifactorial pathophysiology of the disease.

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.

Proposal of a modified scintigraphic method to evaluate duodenogastroesophageal reflux.

Hepatobiliary scintigraphy with 99mTc-HIDA offers a noninvasive method to detect duodenogastric reflux. Biliary reflux was graded using the persistence rather than the intensity of the radioactive refluxate: Grade 0 was considered the absence of reflux, minimal reflux, or reflux in the first 10-15 min; Grade 1 was repetitive reflux lasting less than 10 min; Grade 2 was persistent reflux; and Grade 3 was reflux up to the esophagus. Twenty-five patients with foregut symptoms were studied and results were compared to 24-hr gastric pH monitoring. Scintigraphy and pH monitoring agreed in 15 out of 25 patients (60%), but no correlation was found with the endoscopic findings. The rationale for this approach is based on pathophysiologic evidence that damage to gastric and/or esophageal mucosa is mainly related to the prolonged contact time with duodenal contents. This technique seems to allow a complete functional evaluation of the esophagogastroduodenal tract without causing adjunctive irradiation or discomfort to the patient.

Computerized study with 201T1 of the cold thyroid node.

Because of its physical and potassium-metabolic characteristics 201T1 is more suitable than 131Cs for radioisotopic studies of the cold thyroid nodule, with the further diagnostic possibility of quantitatively assessing intranodular behavior for a specific differentiation among different kinds of neoformations. Using a gamma-camera on line with a computer data processing device, sequential scintiscans were recorded for the first 20-30 min after i.v. administration of 15-20 microCi/kg of radiothallium; delayed sequences were taken at 40-60 min if intranodular uptake appeared. A quantitative appraisal was made of the differential 201T1 uptake-ratio between nodule and healthy thyroid tissue (density-index) and the multiparameter analysis of thyroid time/activity curves generated on the relative regions of interest (ROIs). This computerized study, in 120 out of 293 patients submitted to this radiothallium test, has shown a) diagnostic agreement between clinical-histological and radioisotopic findings in 76 out of 79 colloid-cystic or degenerative neoformations, in all 16 malignant and in 23 out of 25 hyperplastic benign nodules; b) significant statistical difference of the density-index in solid versus cystic but not between benign and malignant nodules; c) different 201T1 kinetics behaviour in different kinds of solid thyroid lesions with a satisfactory statistical difference of the radiothallium nodular disappearance-index.

Scintigraphy of the parathyroid glands with 201TI: experience with 250 operated patients.

Parathyroid scintigraphy confirmed its validity in the preoperative localization of enlarged parathyroids, showing a sensitivity of 82% in a series of 250 patients suffering from primary hyperparathyroidism and successfully operated on. The glands better visualized were in an ectopic site or they were completely or partially outside the thyroid so that they were easily visible without employing digital image subtraction. This is nevertheless necessary to visualize parathyroids in a retrothyroid site but some problems arise, related not only to movements of the patient but also to the instrumentation to perform a correct image subtraction.

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.

[Determination of glomerular filtration during sequential scintigraphy in the evaluation of separate renal function]. / La determinazione del filtrato glomerulare in corso di scintigrafia sequenziale nella valutazione della funzione renale separata.

The serial renal scan, a rapid series of images obtained with a scintillation camera, is explained; it visualizes the arrival to the kidneys and the passage through the parenchyma and the pelvocalyceal system of 99mTc-DTPA, a tracer cleared by glomerular filtration. Dynamic imaging of the kidneys provides more extensive information regarding anatomical structure and renal function that cannot be obtained by other noninvasive techniques. Computerized data analysis provides a lot of parameters, such as global and parenchymal transit times, useful in differentiating obstructive from non-obstructive uropathy, and total and separate glomerular filtration rate. 99mTc-DTPA computer-assisted scintigraphy is recommended as a routine method in nephro-urologic conditions where an accurate evaluation of separate renal function is required.

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.

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.

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.

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.

Isolation of a putative collagen-like gene from the sea urchin Paracentrotus lividus.

Using a Caenorhabditis elegans collagen probe we have isolated a 17.6 kb clone from a Paracentrotus lividus genomic library. Sequencing of nearly 2.6 kb identified five open reading frames flanked at both sides by splice site consensus sequences and coding for ninety-five uninterrupted Gly-X-Y repeats. Interestingly, three of the putative exons exhibit sizes which are identical to those featured by vertebrate fibrillar collagen genes, namely 54 bp and 99 bp. Hybridization of the Gly-X-Y encoding sequences to RNA extracted from different developmental stages identified a specific 6 kb transcript, which appears first at mid-gastrula, greatly increases at prism and then progressively accumulates until pluteus stage. Based on these data, we conclude that the genomic clone is likely to code for a developmentally regulated mRNA whose expression coincides with the reported time of appearance of collagenous molecules in the sea urchin embryo.

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.

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.