T. Hering - Academia.edu (original) (raw)

Papers by T. Hering

Matrix Biology, 2008

Alternative splicing of the type II procollagen gene (COL2A1) is developmentally-regulated during... more Alternative splicing of the type II procollagen gene (COL2A1) is developmentally-regulated during chondrogenesis. Type IIA procollagen (+ exon 2) is synthesized by chondroprogenitor cells while type IIB procollagen (-exon 2) is synthesized by differentiated chondrocytes. Here, we report expression of two additional alternatively spliced COL2A1 isoforms during chondrocyte differentiation of bone marrow derived mesenchymal stem cells (MSCs). One isoform, named IIC, contains only the first 34 nucleotides of exon 2 by use of an alternative 5' splice site, resulting in a premature termination codon and possible nonsense-mediated decay of IIC mRNA. Low levels of the IIC isoform were detected by RT-PCR and Southern analysis of COL2A1 cDNA amplified from differentiating rabbit and human MSCs. A second novel transcript, named IID, arises by use of another 5' alternative splice site in intron 2. The IID isoform contains exon 2 plus 3 nucleotides, resulting in the insertion of an additional amino acid. The IID isoform was co-expressed with the IIA isoform during chondrogenesis, and was approximately one-third as abundant. Deletion of the IIC alternative 5' splice site from a COL2A1 mini-gene construct resulted in a significant increase in the IIA:IIB ratio. A mutant mini-gene that inhibited production of the IID isoform, however, had differential effects on the production of the IIA and IIB isoforms: this was apparently related to the differentiation status of the cell type used. These data suggest that COL2A1 mRNA abundance and other aspects of chondrocyte differentiation may be regulated by the use of these previously undetermined alternative splice sites.

Journal of …, 2005

Purpose: Stem cell-based tissue engineering represents a possible alternative for the repair of c... more Purpose: Stem cell-based tissue engineering represents a possible alternative for the repair of cartilage defects. Both bone marrow and adipose tissue contain pluripotential cells capable of chondrogenesis. This study was a qualitative and quantitative comparison of the ...

Matrix Biology, 2006

mesenchyme, whereas MT2-MMP is more abundant in the epithelium. Blocking of MMP-activity using GM... more mesenchyme, whereas MT2-MMP is more abundant in the epithelium. Blocking of MMP-activity using GM6001 decreases branching and the activity of MMP2, then we used siRNA to knockdown MMP2, MT1-and MT3-MMP, and we found that targeting MMP2 and MT1-MMP decreases branching more than MT3-MMP knockdown. Using gelatin zymography, we found that the MMP2-activation decreases using MMP2 and MT1-but not MT3-MMP siRNA. In summary: 1. Soluble MMPs 2, 9 and 11, and membrane-type MT1-, MT2and MT3-MMP are highly expressed and have discrete localization during SMG morphogenesis; 2. MMP2 and MT1-MMP targeted by siRNA are more effective to reduce SMG branching than MT3-MMP; 3. SMG morphogenesis may be compensated by MT3-MMP or other MMP-like proteases after MMP2 and MT1-MMP siRNA knockdown.

Osteoarthritis and Cartilage, 1995

The proteoglycans synthesized by human osteoarthritic femoral head cartilage and nonarthritic art... more The proteoglycans synthesized by human osteoarthritic femoral head cartilage and nonarthritic articular cartilage age-matched to the osteoarthritic cartilage specimens was studied in explant cultures and in chondrocytes generated by explant outgrowth from the cartilages. Twenty-four hours after explanation, both nonarthritic articular cartilage and osteoarthritic cartilage synthesized principally one large proteoglycan core protein that migrated on 3-5% acrylamide gels with an apparent molecular mass (Mr) of ~520kDa after enzymatic digestion with chondroitinase ABC and keratanase. The proteoglycan was found in both the explant itself and in the medium compartment of the culture as well. This proteoglycan contained chondroitin-6-sulfate, keratan sulfate and the hyaluronan binding region as evidenced by immunoblotting with murine anti-proteoglycan monoclonal antibodies indicating that the proteoglycan was aggrecan. To a much lesser extent two additional proteoglycan core proteins were also found in the explant but were not seen in the culture medium compartment. These proteoglycans possessed apparent Mr's of ~ 480 kDa and ~ 390 kDa on 3-5% acrylamide gels after chondroitinase ABC and keratanase digestion. The medium compartment contained principally the 520 kDa proteoglycan core protein. In osteoarthritic cartilage explants, the pattern of newly synthesized proteoglycans recovered from the tissue as assessed on 3-16% polyacrylamide gradient gels remained relatively the same from day 1 after explantation up to 36 days of culture. By contrast, the proteoglycans recovered from the culture medium contained chondroitin sulfate and keratan sulfate after 1, 7 and 21 days in culture but by 36 days appeared to contain only chondroitin sulfate. Chondrocytes generated from osteoarthritic cartilage and age-matched nonarthritic articular cartilage synthesized different patterns of large (greater than 200 kDa) proteoglycan. Whereas chondrocytes derived from osteoarthritic cartilage continued to synthesize principally the ~ 520 kDa proteoglycan core protein, the chondrocytes derived from nonarthritic cartilage synthesized in addition to this proteoglycan, abundant amounts of the other two proteoglycan core proteins as well.

Archives of Biochemistry and Biophysics, 1994

Experimental Cell Research, 1998

cartilage when implanted in vivo . A culture sys-A culture system that facilitates the chondrogen... more cartilage when implanted in vivo . A culture sys-A culture system that facilitates the chondrogenic tem has been developed in which these cells will undifferentiation of rabbit bone marrow-derived mesen-dergo osteogenic differentiation in vitro . However, chymal progenitor cells has been developed. Cells obattempts to develop in vitro conditions in which mesentained in bone marrow aspirates were first isolated by chymal progenitor cells isolated from postnatal mammonolayer culture and then transferred into tubes and malian bone marrow will progress down the chondroallowed to form three-dimensional aggregates in a genic lineage have been less successful. There are studchemically defined medium. The inclusion of 10 07 M ies reporting in vitro chondrogenesis using postnatal dexamethasone in the medium induced chondrogenic mammalian cells , but none have demonstrated differentiation of cells within the aggregate as evihistologically identifiable cartilage formation, although denced by the appearance of toluidine blue metachrotype II collagen production has been detected, sugmasia and the immunohistochemical detection of type gesting at least prechondroid tissue production.

Matrix Biology, 2008

Alternative splicing of the type II procollagen gene (COL2A1) is developmentally-regulated during... more Alternative splicing of the type II procollagen gene (COL2A1) is developmentally-regulated during chondrogenesis. Type IIA procollagen (+ exon 2) is synthesized by chondroprogenitor cells while type IIB procollagen (-exon 2) is synthesized by differentiated chondrocytes. Here, we report expression of two additional alternatively spliced COL2A1 isoforms during chondrocyte differentiation of bone marrow derived mesenchymal stem cells (MSCs). One isoform, named IIC, contains only the first 34 nucleotides of exon 2 by use of an alternative 5' splice site, resulting in a premature termination codon and possible nonsense-mediated decay of IIC mRNA. Low levels of the IIC isoform were detected by RT-PCR and Southern analysis of COL2A1 cDNA amplified from differentiating rabbit and human MSCs. A second novel transcript, named IID, arises by use of another 5' alternative splice site in intron 2. The IID isoform contains exon 2 plus 3 nucleotides, resulting in the insertion of an additional amino acid. The IID isoform was co-expressed with the IIA isoform during chondrogenesis, and was approximately one-third as abundant. Deletion of the IIC alternative 5' splice site from a COL2A1 mini-gene construct resulted in a significant increase in the IIA:IIB ratio. A mutant mini-gene that inhibited production of the IID isoform, however, had differential effects on the production of the IIA and IIB isoforms: this was apparently related to the differentiation status of the cell type used. These data suggest that COL2A1 mRNA abundance and other aspects of chondrocyte differentiation may be regulated by the use of these previously undetermined alternative splice sites.

Journal of …, 2005

Purpose: Stem cell-based tissue engineering represents a possible alternative for the repair of c... more Purpose: Stem cell-based tissue engineering represents a possible alternative for the repair of cartilage defects. Both bone marrow and adipose tissue contain pluripotential cells capable of chondrogenesis. This study was a qualitative and quantitative comparison of the ...

Matrix Biology, 2006

mesenchyme, whereas MT2-MMP is more abundant in the epithelium. Blocking of MMP-activity using GM... more mesenchyme, whereas MT2-MMP is more abundant in the epithelium. Blocking of MMP-activity using GM6001 decreases branching and the activity of MMP2, then we used siRNA to knockdown MMP2, MT1-and MT3-MMP, and we found that targeting MMP2 and MT1-MMP decreases branching more than MT3-MMP knockdown. Using gelatin zymography, we found that the MMP2-activation decreases using MMP2 and MT1-but not MT3-MMP siRNA. In summary: 1. Soluble MMPs 2, 9 and 11, and membrane-type MT1-, MT2and MT3-MMP are highly expressed and have discrete localization during SMG morphogenesis; 2. MMP2 and MT1-MMP targeted by siRNA are more effective to reduce SMG branching than MT3-MMP; 3. SMG morphogenesis may be compensated by MT3-MMP or other MMP-like proteases after MMP2 and MT1-MMP siRNA knockdown.

Osteoarthritis and Cartilage, 1995

The proteoglycans synthesized by human osteoarthritic femoral head cartilage and nonarthritic art... more The proteoglycans synthesized by human osteoarthritic femoral head cartilage and nonarthritic articular cartilage age-matched to the osteoarthritic cartilage specimens was studied in explant cultures and in chondrocytes generated by explant outgrowth from the cartilages. Twenty-four hours after explanation, both nonarthritic articular cartilage and osteoarthritic cartilage synthesized principally one large proteoglycan core protein that migrated on 3-5% acrylamide gels with an apparent molecular mass (Mr) of ~520kDa after enzymatic digestion with chondroitinase ABC and keratanase. The proteoglycan was found in both the explant itself and in the medium compartment of the culture as well. This proteoglycan contained chondroitin-6-sulfate, keratan sulfate and the hyaluronan binding region as evidenced by immunoblotting with murine anti-proteoglycan monoclonal antibodies indicating that the proteoglycan was aggrecan. To a much lesser extent two additional proteoglycan core proteins were also found in the explant but were not seen in the culture medium compartment. These proteoglycans possessed apparent Mr's of ~ 480 kDa and ~ 390 kDa on 3-5% acrylamide gels after chondroitinase ABC and keratanase digestion. The medium compartment contained principally the 520 kDa proteoglycan core protein. In osteoarthritic cartilage explants, the pattern of newly synthesized proteoglycans recovered from the tissue as assessed on 3-16% polyacrylamide gradient gels remained relatively the same from day 1 after explantation up to 36 days of culture. By contrast, the proteoglycans recovered from the culture medium contained chondroitin sulfate and keratan sulfate after 1, 7 and 21 days in culture but by 36 days appeared to contain only chondroitin sulfate. Chondrocytes generated from osteoarthritic cartilage and age-matched nonarthritic articular cartilage synthesized different patterns of large (greater than 200 kDa) proteoglycan. Whereas chondrocytes derived from osteoarthritic cartilage continued to synthesize principally the ~ 520 kDa proteoglycan core protein, the chondrocytes derived from nonarthritic cartilage synthesized in addition to this proteoglycan, abundant amounts of the other two proteoglycan core proteins as well.

Archives of Biochemistry and Biophysics, 1994

Experimental Cell Research, 1998

cartilage when implanted in vivo . A culture sys-A culture system that facilitates the chondrogen... more cartilage when implanted in vivo . A culture sys-A culture system that facilitates the chondrogenic tem has been developed in which these cells will undifferentiation of rabbit bone marrow-derived mesen-dergo osteogenic differentiation in vitro . However, chymal progenitor cells has been developed. Cells obattempts to develop in vitro conditions in which mesentained in bone marrow aspirates were first isolated by chymal progenitor cells isolated from postnatal mammonolayer culture and then transferred into tubes and malian bone marrow will progress down the chondroallowed to form three-dimensional aggregates in a genic lineage have been less successful. There are studchemically defined medium. The inclusion of 10 07 M ies reporting in vitro chondrogenesis using postnatal dexamethasone in the medium induced chondrogenic mammalian cells , but none have demonstrated differentiation of cells within the aggregate as evihistologically identifiable cartilage formation, although denced by the appearance of toluidine blue metachrotype II collagen production has been detected, sugmasia and the immunohistochemical detection of type gesting at least prechondroid tissue production.