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Collagen IIB mRNA levels and chondrogenesis are enhanced by functional suppression of retinoic acid receptor in limb bud cells
376
Fifth International Conference on the Molecular Biology and Pathology of Matrix
for HDMVEC culture. In contrast to the maximal expression of type I and VI collagens and FN detected in HDMVEC in the absence of growth factors, aFGF decreased mRNA levels for type I collagen by 43%, for type VI collagen by 52% and for FN by 47%. The decreases in mRNA levels caused by bFGF were 37%, 41% and 36% respectively. Heparin by itself was capable of suppressing the expression of the collagen and FN genes. However, FGFs potentiated the negative effect of heparin on ECM gene expression. Thus, our data demonstrate that heparin, ECGF, aFGF and bFGF regulate ECM gene expression in HDMVEC in vitro, and suggest that these growth factors may modulate the expression of matrix genes in vivo. Altered expression of ECM genes by HDMVEC may play important role in diseases affecting the microvasculature such as systemic sclerosis, diabetes mellitus, and arteriosclerosis.
in all of the ulcer edge biopsies but less frequently in the ulcer base biopsies. IL-10t transcripts were detected in only two biopsies, both from the ulcer base regions. These data demonstrate spatially differential expression of collagens I and III in lower limb ulcers, collagen I being expressed predominantly at the ulcer edge and collagen III in the ulcer base. This is consistent with the hypothesis that ulcer healing is more advanced at the ulcer edge than the ulcer base. IL-113 expression was detected in all areas of the ulcers and at all time points, whereas IL-10~ transcripts were only detected in the ulcer-base region. The major cellular sources of IL-la and 13are keratinocytes and monocytic cells, but as the ulcer base is denuded of keratinocytes the ILs at this site are likely to be predominantly monocyte in origin. IL-10t depletion impairs healing in model systems and augmcntation of IL-10t accelerates healing with the restoration of normal dermal architecture. It is possible that IL-10t depletion in the ulcer base contributes towards the chronicity of leg ulcers.
Collagen and Cytokine Gene Expression in Lower Limb Ulcers in Humans I. Hopkinson, I. Anglin, R.J. Whiston and K.G. Harding
Wound HealingResearch Unit, Wound Healing Research Unit, University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XN, UK Lower limb ulceration is a significant cause of misery and morbidity (incidence 2-6% in over-60year olds in the UK). The co-ordinated expression of collagens I and III which is essential for dermal healing has been demonstrated to be disrupted in these lesions. There is some evidence that interleukin 1 regulates the expression of fibrillar collagens and is required for normal dermal repair. The aim of this study was to analyse the expression of collagens I and III and interleukins 10t and 113in lower limb ulcers. Punch biopsies were obtained from the edge of 12 lower limb ulcers at various stages of healing in 12 patients and excision biopsies obtained from four leg ulcers from amputated limbs (Ethical committee approved. Informed consent obtained). Morphological analysis was performed on sections from the excision biopsies and RNA was isolated from all the biopsies. The expression fo CollA1, Co13A1 IL-10t and IL-113 was analyzed using RT-PCR. Collagen I was distributed mostly at the edge of the ulcers and in the intact collagenous fascial layer. Collagen III was distributed in a perivascular pattern and in the central base regions of the ulcers. Collagen III and IL-113 transcripts were detected in biopsies from edge and base regions of the ulcers at all time points examined. Collagen I transcripts were detected
Collagen IIB m R N A Levels and Chondrogenesis are E n h a n c e d by F u n c t i o n a l Suppression of Retinoic Acid R e c e p t o r in L i m b B u d Cells H. Jiang, D.R. Soprano, K.J. Soprano and D.M. Kochhar Department of Anatomy and Developmental Biology, Thomas Jefferson University;and Departments of Biochemistry and Microbiology, Temple University, Philadelphia, PA, USA The objective is to study gene control over normal spatial patterning of skeletal elements in embryonic limb bud development. Since exogenously supplied retinoic acid (RA), a metabolite of vitamin A, influences this pattern and produces chondrodysplastic anomalies, here we investigated if the forced absence of specific nuclear retinoic acid receptors (RAR) would block the teratogenic effects of RA. Replicate "micromass" cultures of mouse limb bud cells were exposed to either RA alone (3-300 nM) or RA plus 20-80 gg/ml of antisense deoxyoligonucleotides directed at the 5' end of RAR-~2 mRNA. Chondrogenesis was monitored by the 1) relative abundance of collagen liB (without exon 2) versus type IIA mRNA in the differentiating cells, 2) synthesis of 35S-labeled proteoglycans, and 3) number and morphology of cartilage nodules. Chondrogenesis was inhibited by RA in a dose-dependent manner where 50% inhibition
Fifth International Conference on the Molecular Biology and Pathology of Matrix occurred at 50 _+ 15 nM RA. The antisense probe ameliorated the effect of RA on chondrogenesis in a dose-dependent manner, where 80 mg/ml abolished almost completely the inhibitory effects of 300 nM RA. The results imply that RAR-[32 is directly involved in teratogenic events. Further pathway of action still remains to be resolved. Surprisingly, the antisense RAR-]32 probe alone significantly enhanced all end-points of chondrogenic cell differentiation in normal control limb bud cells, indicating that the endogenous receptor serves to keep the cells in an undifferentiated state in the normal developmental program. (Supported by NIH and March of Dimes)
377
Patterns of Collagen Expression in Ovarian Tumors S. Kauppila, J. Saarela, J. Risteli, F. Stenbfick, A. Kauppila and L. Risteli Departments of Medical Biochemist-y,Clinical Chemistry, Pathology and Gynecology, University of Oulu, Oulu, Finland
We have previously shown that very high concentrations of the propeptide domains of type I and type III procollagens are found both in the tumor cysts and in peritoneal ascitic fluid of patients with ovarian cancer (Cancer Res 53" 5028-5032, 1993), indicating that a strong fibroproliferative response is induced by these tumors locally and further away in the peritoneal cavity. The N-terminal propeptide of type III Okadaic Acid Suppresses Type I Collagen procollagen (PIIINP) in serum functions as a biochemGene Expression at Transcriptional Level in ical marker of this process (Cancer Res 49:18851889, 1989). Here we wanted to find out whether the Cultured Fibroblasts malignant cells or stromal fibroblasts are responsible for the increased collagen production in ovarian V.-M. K~ih~iri, J. Westermarck and E. Ilvonen tumors. Formalin-fixed, paraffin-embedded tissue samples were studied by in situ hybridization. ConDepartments of Dermatology, Medical Biochemistry, and MediCity Research Laboratory, University of Turku, FINstructs containing fragments of cDNAs for the human 20520 Turku, Finland pro00(I) and pro0tl(III) chains in the pGEM1 vector were kind gifts of Eero Vuorio (University of Turku, Type I collagen is an abundant collagen of human Finland). A 571-base long fragment was cut from connective tissues, including dermis. We have cDNA for the human pro0t2(I)chain and subcloned examined the effect of okadaic acid (OA), an inhibitor into pBluescript SK(-) vector. Sense and anti-sense of serine/threonine-specific protein phosphatases 1 RNA probes labeled with S3s were prepared with SP6, and 2A, on type I collagen gene expression in normal T7 and T3 RNA polymerases. The hybridization human skin fibroblasts and mouse NIH-3T3 cells. reactions were visualized by autoradiography. Strong signals for both chains of type I procollagen Treatment of human skin fibroblasts with OA (20 ng/ml) for 24 h markedly reduced pro0~l(I) and were seen in stromal fibroblasts around the cystic pro0t2(I) collagen mRNA levels. This effect was not structures containing neoplastic cells in well differendependent on protein synthesis and was not affected tiated ovarian adenocarcinomas and in Brenner by simultaneous treatment with dexamethasone or tumors. Such a reaction was not seen around benign retinoic acid (both 1 p.M). Treatment of fibroblasts ovarian cysts. In poorly differentiated tumors strong with OA in combination with transforming growth signals were also seen occasionally in the neoplastic factor-J3 (TGF-~3) entirely abolished the TGF-~3-elicited cells themselves. In these cases, the codistribution of enhancement of type I procollagen mRNA levels. In the signals for the prootl(I) and pro0t2(I) chains was transiently transfected NIH-3T3 cells OA suppressed not always complete. The sense controls were negathe activity of a 3.5-kb human pro0t2(I) collagen tive for all these findings. promoter/CAT construct in a dose-dependent manner, The results suggest that the production of type I the m a x i m a l i n h i b i t i o n (70%) noted with procollagen in well-differentiated ovarian cancers is a concentration 80 ng/ml. These results indicate that function of stromal fibroblasts, but that in poorly difprotein phosphatases 1 and 2A have an important role ferentiated tumors there is also aberrant expression of as positive regulators of type I collagen gene this procollagen or one of its component chains in the expression. The results also suggest that elucidation neoplastic cells. of the molecular m e c h a n i s m of OA-elicited suppression of type I collagen gene expression may be useful in development of more targeted therapeutic modalities for fibrotic disorders, such as scleroderma, keloids, liver cirrhosis and pulmonary fibrosis.
Fifth International Conference on the Molecular Biology and Pathology of Matrix
for HDMVEC culture. In contrast to the maximal expression of type I and VI collagens and FN detected in HDMVEC in the absence of growth factors, aFGF decreased mRNA levels for type I collagen by 43%, for type VI collagen by 52% and for FN by 47%. The decreases in mRNA levels caused by bFGF were 37%, 41% and 36% respectively. Heparin by itself was capable of suppressing the expression of the collagen and FN genes. However, FGFs potentiated the negative effect of heparin on ECM gene expression. Thus, our data demonstrate that heparin, ECGF, aFGF and bFGF regulate ECM gene expression in HDMVEC in vitro, and suggest that these growth factors may modulate the expression of matrix genes in vivo. Altered expression of ECM genes by HDMVEC may play important role in diseases affecting the microvasculature such as systemic sclerosis, diabetes mellitus, and arteriosclerosis.
in all of the ulcer edge biopsies but less frequently in the ulcer base biopsies. IL-10t transcripts were detected in only two biopsies, both from the ulcer base regions. These data demonstrate spatially differential expression of collagens I and III in lower limb ulcers, collagen I being expressed predominantly at the ulcer edge and collagen III in the ulcer base. This is consistent with the hypothesis that ulcer healing is more advanced at the ulcer edge than the ulcer base. IL-113 expression was detected in all areas of the ulcers and at all time points, whereas IL-10~ transcripts were only detected in the ulcer-base region. The major cellular sources of IL-la and 13are keratinocytes and monocytic cells, but as the ulcer base is denuded of keratinocytes the ILs at this site are likely to be predominantly monocyte in origin. IL-10t depletion impairs healing in model systems and augmcntation of IL-10t accelerates healing with the restoration of normal dermal architecture. It is possible that IL-10t depletion in the ulcer base contributes towards the chronicity of leg ulcers.
Collagen and Cytokine Gene Expression in Lower Limb Ulcers in Humans I. Hopkinson, I. Anglin, R.J. Whiston and K.G. Harding
Wound HealingResearch Unit, Wound Healing Research Unit, University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XN, UK Lower limb ulceration is a significant cause of misery and morbidity (incidence 2-6% in over-60year olds in the UK). The co-ordinated expression of collagens I and III which is essential for dermal healing has been demonstrated to be disrupted in these lesions. There is some evidence that interleukin 1 regulates the expression of fibrillar collagens and is required for normal dermal repair. The aim of this study was to analyse the expression of collagens I and III and interleukins 10t and 113in lower limb ulcers. Punch biopsies were obtained from the edge of 12 lower limb ulcers at various stages of healing in 12 patients and excision biopsies obtained from four leg ulcers from amputated limbs (Ethical committee approved. Informed consent obtained). Morphological analysis was performed on sections from the excision biopsies and RNA was isolated from all the biopsies. The expression fo CollA1, Co13A1 IL-10t and IL-113 was analyzed using RT-PCR. Collagen I was distributed mostly at the edge of the ulcers and in the intact collagenous fascial layer. Collagen III was distributed in a perivascular pattern and in the central base regions of the ulcers. Collagen III and IL-113 transcripts were detected in biopsies from edge and base regions of the ulcers at all time points examined. Collagen I transcripts were detected
Collagen IIB m R N A Levels and Chondrogenesis are E n h a n c e d by F u n c t i o n a l Suppression of Retinoic Acid R e c e p t o r in L i m b B u d Cells H. Jiang, D.R. Soprano, K.J. Soprano and D.M. Kochhar Department of Anatomy and Developmental Biology, Thomas Jefferson University;and Departments of Biochemistry and Microbiology, Temple University, Philadelphia, PA, USA The objective is to study gene control over normal spatial patterning of skeletal elements in embryonic limb bud development. Since exogenously supplied retinoic acid (RA), a metabolite of vitamin A, influences this pattern and produces chondrodysplastic anomalies, here we investigated if the forced absence of specific nuclear retinoic acid receptors (RAR) would block the teratogenic effects of RA. Replicate "micromass" cultures of mouse limb bud cells were exposed to either RA alone (3-300 nM) or RA plus 20-80 gg/ml of antisense deoxyoligonucleotides directed at the 5' end of RAR-~2 mRNA. Chondrogenesis was monitored by the 1) relative abundance of collagen liB (without exon 2) versus type IIA mRNA in the differentiating cells, 2) synthesis of 35S-labeled proteoglycans, and 3) number and morphology of cartilage nodules. Chondrogenesis was inhibited by RA in a dose-dependent manner where 50% inhibition
Fifth International Conference on the Molecular Biology and Pathology of Matrix occurred at 50 _+ 15 nM RA. The antisense probe ameliorated the effect of RA on chondrogenesis in a dose-dependent manner, where 80 mg/ml abolished almost completely the inhibitory effects of 300 nM RA. The results imply that RAR-[32 is directly involved in teratogenic events. Further pathway of action still remains to be resolved. Surprisingly, the antisense RAR-]32 probe alone significantly enhanced all end-points of chondrogenic cell differentiation in normal control limb bud cells, indicating that the endogenous receptor serves to keep the cells in an undifferentiated state in the normal developmental program. (Supported by NIH and March of Dimes)
377
Patterns of Collagen Expression in Ovarian Tumors S. Kauppila, J. Saarela, J. Risteli, F. Stenbfick, A. Kauppila and L. Risteli Departments of Medical Biochemist-y,Clinical Chemistry, Pathology and Gynecology, University of Oulu, Oulu, Finland
We have previously shown that very high concentrations of the propeptide domains of type I and type III procollagens are found both in the tumor cysts and in peritoneal ascitic fluid of patients with ovarian cancer (Cancer Res 53" 5028-5032, 1993), indicating that a strong fibroproliferative response is induced by these tumors locally and further away in the peritoneal cavity. The N-terminal propeptide of type III Okadaic Acid Suppresses Type I Collagen procollagen (PIIINP) in serum functions as a biochemGene Expression at Transcriptional Level in ical marker of this process (Cancer Res 49:18851889, 1989). Here we wanted to find out whether the Cultured Fibroblasts malignant cells or stromal fibroblasts are responsible for the increased collagen production in ovarian V.-M. K~ih~iri, J. Westermarck and E. Ilvonen tumors. Formalin-fixed, paraffin-embedded tissue samples were studied by in situ hybridization. ConDepartments of Dermatology, Medical Biochemistry, and MediCity Research Laboratory, University of Turku, FINstructs containing fragments of cDNAs for the human 20520 Turku, Finland pro00(I) and pro0tl(III) chains in the pGEM1 vector were kind gifts of Eero Vuorio (University of Turku, Type I collagen is an abundant collagen of human Finland). A 571-base long fragment was cut from connective tissues, including dermis. We have cDNA for the human pro0t2(I)chain and subcloned examined the effect of okadaic acid (OA), an inhibitor into pBluescript SK(-) vector. Sense and anti-sense of serine/threonine-specific protein phosphatases 1 RNA probes labeled with S3s were prepared with SP6, and 2A, on type I collagen gene expression in normal T7 and T3 RNA polymerases. The hybridization human skin fibroblasts and mouse NIH-3T3 cells. reactions were visualized by autoradiography. Strong signals for both chains of type I procollagen Treatment of human skin fibroblasts with OA (20 ng/ml) for 24 h markedly reduced pro0~l(I) and were seen in stromal fibroblasts around the cystic pro0t2(I) collagen mRNA levels. This effect was not structures containing neoplastic cells in well differendependent on protein synthesis and was not affected tiated ovarian adenocarcinomas and in Brenner by simultaneous treatment with dexamethasone or tumors. Such a reaction was not seen around benign retinoic acid (both 1 p.M). Treatment of fibroblasts ovarian cysts. In poorly differentiated tumors strong with OA in combination with transforming growth signals were also seen occasionally in the neoplastic factor-J3 (TGF-~3) entirely abolished the TGF-~3-elicited cells themselves. In these cases, the codistribution of enhancement of type I procollagen mRNA levels. In the signals for the prootl(I) and pro0t2(I) chains was transiently transfected NIH-3T3 cells OA suppressed not always complete. The sense controls were negathe activity of a 3.5-kb human pro0t2(I) collagen tive for all these findings. promoter/CAT construct in a dose-dependent manner, The results suggest that the production of type I the m a x i m a l i n h i b i t i o n (70%) noted with procollagen in well-differentiated ovarian cancers is a concentration 80 ng/ml. These results indicate that function of stromal fibroblasts, but that in poorly difprotein phosphatases 1 and 2A have an important role ferentiated tumors there is also aberrant expression of as positive regulators of type I collagen gene this procollagen or one of its component chains in the expression. The results also suggest that elucidation neoplastic cells. of the molecular m e c h a n i s m of OA-elicited suppression of type I collagen gene expression may be useful in development of more targeted therapeutic modalities for fibrotic disorders, such as scleroderma, keloids, liver cirrhosis and pulmonary fibrosis.