History of the Department of Biochemistry and Molecular Biology

This departmental history was written on the occasion of the UND Quasquicentennial in 2008.https://commons.und.edu/departmental-histories/1063/thumbnail.jp

Proapoptotic fibronectin fragment induces the degradation of ubiquitinated p53 via proteasomes in periodontal ligament cells

Ghosh A, Joo NE, Chen TC, Kapila YL. Proapoptotic fibronectin fragment induces the degradation of ubiquitinated p53 via proteasomes in periodontal ligament cells. J Periodont Res 2010; 45: 481–487. © 2010 John Wiley & Sons A/S The extracellular matrix (ECM) plays a key role in signaling necessary for tissue remodeling and cell survival. However, signals from the ECM altered by disease, e.g. inflammatory diseases such as periodontitis and arthritis, may lead to apoptosis or programmed cell death of resident cells. Previously, we found that a disease-associated fibronectin fragment triggers apoptosis of primary human periodontal ligament cells via a novel apoptotic pathway in which the tumor suppressor, p53, is transcriptionally downregulated.We used immunofluorescence, transfection assays, western blotting and ELISAs to show that p53 is degraded by a proteasomal pathway in response to a proapoptotic disease-associated fibronectin fragment.We found that in these apoptotic conditions, p53 is further downregulated by post-translational ubiquitination and subsequent targeting to proteasomes for degradation. Pretreatment of cells with the proteasomal inhibitors MG132 and lactacystin rescued the cells from apoptosis. The p53 levels in cells transfected with ubiquitin small interfering RNA were resistant to degradation induced by the proapoptotic fibronectin fragment, showing that ubiquitination is important for the proapoptotic fibronectin fragment-induced degradation of p53.These data show that a proapoptotic fibronectin matrix induces ubiquitination and degradation of p53 in the proteasome as part of a novel mechanism of apoptosis associated with inflammatory diseases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79162/1/j.1600-0765.2009.01261.x.pd

The cartilage chondrolytic mechanism of fibronectin fragments involves MAP kinases: comparison of three fragments and native fibronectin

SummaryObjectiveTo define the role of mitogen activated protein (MAP) kinases in fibronectin fragment (Fn-f) mediated matrix metalloproteinase (MMP) upregulation and damage to bovine cartilage and to compare activities of three Fn-fs with native fibronectin (Fn), which is inactive in terms of cartilage damage.MethodsBovine chondrocytes were cultured with three Fn-fs, an amino-terminal 29-kDa, a gelatin-binding 50-kDa and a central 140-kDa Fn-f or native Fn at concentrations from 0.01 to 1μM, concentrations lower than those found in osteoarthritis synovial fluids. Lysates were probed for activation of MAP kinases, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and stress activated protein kinase/c-jun N-terminal kinase (SAPK/JNK). Confocal fluorescent microscopy was used to visualize movement of activated kinases. Kinase inhibitors were tested for their abilities to block Fn-f mediated protein upregulation of MMP-3 and MMP-13 and Fn-f induced depletion of cartilage proteoglycan (PG) from cultured explants.ResultsThe 29-kDa, the most potent Fn-f in terms of cartilage damage, enhanced phosphorylation of ERK1/2, p38 and JNK1/2 within a 1-h incubation while the 50 and 140-kDa Fn-fs required up to 4h for maximal activity and native Fn was only minimally active toward p38 and JNK, but did strongly activate ERK1/2. The activated kinases displayed a distribution toward the nuclear membrane and within the nucleus. MAP kinase inhibitors markedly decreased Fn-f mediated upregulation of MMP-3 or MMP-13 and Fn-f mediated cartilage PG depletion.ConclusionsThese results suggest that Fn-fs upregulate MMP-3 and MMP-13 in bovine chondrocytes through MAP kinases and that kinase inhibitors afford protection against this degenerative pathway

Biomechanical modulation of collagen fragment-induced anabolic and catabolic activities in chondrocyte/agarose constructs

The present study examined the effect of collagen fragments on anabolic and catabolic activities by chondrocyte/agarose constructs subjected to dynamic compression..

Studies on the Ultrastructure of Fibrin Lacking Fibrinopeptide B (β-Fibrin)

Release of fibrinopeptide B from fibrinogen by copperhead venom procoagulant enzyme results in a form of fibrin (beta-fibrin) with weaker self-aggregation characteristics than the normal product (alpha beta-fibrin) produced by release of fibrinopeptides A (FPA) and B (FPB) by thrombin. We investigated the ultrastructure of these two types of fibrin as well as that of beta-fibrin prepared from fibrinogen Metz (A alpha 16 Arg----Cys), a homozygous dysfibrinogenemic mutant that does not release FPA. At 14 degrees C and physiologic solvent conditions (0.15 mol/L of NaCl, 0.015 mol/L of Tris buffer pH 7.4), the turbidity (350 nm) of rapidly polymerizing alpha beta-fibrin (thrombin 1 to 2 U/mL) plateaued in less than 6 min and formed a “coarse” matrix consisting of anastomosing fiber bundles (mean diameter 92 nm). More slowly polymerizing alpha beta-fibrin (thrombin 0.01 and 0.001 U/mL) surpassed this turbidity after greater than or equal to 60 minutes and concomitantly developed a network of thicker fiber bundles (mean diameters 118 and 186 nm, respectively). Such matrices also contained networks of highly branched, twisting, “fine” fibrils (fiber diameters 7 to 30 nm) that are usually characteristic of matrices formed at high ionic strength and pH. Slowly polymerizing beta-fibrin, like slowly polymerizing alpha beta-fibrin, displayed considerable quantities of fine matrix in addition to an underlying thick cable network (mean fiber diameter 135 nm), whereas rapidly polymerizing beta-fibrin monomer was comprised almost exclusively of wide, poorly anastomosed, striated cables (mean diameter 212 nm). Metz beta-fibrin clots were more fragile than those of normal beta-fibrin and were comprised almost entirely of a fine network. Metz fibrin could be induced, however, to form thick fiber bundles (mean diameter 76 nm) in the presence of albumin at a concentration (500 mumol/L) in the physiologic range and resembled a Metz plasma fibrin clot in that regard. The diminished capacity of Metz beta-fibrin to form thick fiber bundles may be due to impaired use or occupancy of a polymerization site exposed by FPB release. Our results indicate that twisting fibrils are an inherent structural feature of all forms of assembling fibrin, and suggest that mature beta-fibrin or alpha beta-fibrin clots develop from networks of thin fibrils that have the ability to coalesce to form thicker fiber bundles

Mathematical modelling of cytokines, MMPs and fibronectin fragments in osteoarthritic cartilage

Osteoarthritis (OA) is a degenerative disease which causes pain and stiffness in joints. OA progresses through excessive degradation of joint cartilage, eventually leading to significant joint degeneration and loss of function. Cytokines, a group of cell signalling proteins, present in raised concentrations in OA joints, can be classified into pro-inflammatory and anti-inflammatory groups. They mediate cartilage degradation through several mechanisms, primarily the up-regulation of matrix metalloproteinases (MMPs), a group of collagen-degrading enzymes. In this paper we show that the interactions of cytokines within cartilage have a crucial role to play in OA progression and treatment. We develop a four-variable ordinary differential equation model for the interactions between pro- and anti-inflammatory cytokines, MMPs and fibronectin fragments (Fn-fs), a by-product of cartilage degradation and upregulator of cytokines. We show that the model has four classes of dynamic behaviour: homoeostasis, bistable inflammation, tristable inflammation and persistent inflammation. We show that positive and negative feedbacks controlling cytokine production rates can determine either a pre-disposition to OA or initiation of OA. Further, we show that manipulation of cytokine, MMP and Fn-fs levels can be used to treat OA, but we suggest that multiple treatment targets may be essential to halt or slow disease progression

Fibronectin III 13-14 Domains Induce Joint Damage via Toll-Like Receptor 4 Activation and Synergize with Interleukin-1 and Tumour Necrosis Factor

Cartilage loss is a feature of chronic arthritis. It results from degradation of the extracellular matrix which is composed predominantly of aggrecan and type II collagen. Extracellular matrix degradation is mediated by aggrecanases and matrix metalloproteinases (MMPs). Recently, a number of endogenous matrix molecules, including fibronectin (FN), have been implicated in mediating cartilage degradation. We were interested in studying the C-terminal heparin-binding region of FN since it mediates aggrecan and type II collagen breakdown in cartilage, but the specific FN domains responsible for proteolytic enzyme activity and their receptors in cartilage are unknown. In this study, the ability of recombinant FN domains to induce cartilage breakdown was tested. We found that the FN III 13-14 domains in the C-terminal heparin-binding region of FN are potent inducers of aggrecanase activity in articular cartilage. In murine studies, the FN III 13-14-induced aggrecanase activity was inhibited in Toll-like receptor 4 (TLR4) knockout mice but not wild-type mice. FN III 13-14 domains also synergized with the known catabolic cytokines interleukin-1α and tumour necrosis factor and induced secretion of MMP-1, MMP-3, gp38 and serum amyloid-like protein A in chondrocytes. Our studies provide a mechanistic link between the innate immune receptor TLR4 and sterile arthritis induced by the FN III 13-14 domains of the endogenous matrix molecule FN