Mario C. Rico

Expression and function of periostin-isoforms in bone.

Periostin was originally identified in MC3T3‐E1 osteoblast‐like cells. We have identified an isoform of periostin referred to as periostin‐like‐factor (PLF). It is homologous to other proteins such as fasciclin I (fas I), MPB70, βIG‐H3, and Algal‐CAMs. All of these proteins are implicated in regulating cell adhesion. PLF and the other isoforms of periostin differ in their C‐terminal sequences. PLF and periostin differ in two specific regions, between 673 and 699 amino acids (aa) and 785–812 aa. Periostin isoforms are expressed in vivo and in vitro during the stages of osteoblast differentiation and maturation. Their mRNAs are present in pre‐osteoblast cells as detected by in situ hybridization, and the proteins are between 86 and 93 kD in size as determined by Western blot analysis. Antisense oligonucleotides and antibodies directed against the isoforms of periostin were used to block the activity of these proteins. In both cases, the levels of osteoblast‐specific‐differentiation markers were markedly reduced suggesting a role for these proteins in osteoblast differentiation.

Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts.

Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N‐linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3‐ to 4‐fold overexpressed in op versus normal bone. RT‐PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non‐osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast‐specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function. J. Cell. Biochem. 84: 12–26, 2002.

Osteoactivin, an anabolic factor that regulates osteoblast differentiation and function

Osteoactivin (OA) is a novel glycoprotein that is highly expressed during osteoblast differentiation. Using Western blot analysis, our data show that OA protein has two isoforms, one is transmembranous and the other is secreted into the conditioned medium of primary osteoblasts cultures. Fractionation of osteoblast cell compartments showed that the mature, glycosylated OA isoform of 115 kDa is found in the membranous fraction. Both OA isoforms (secreted and transmembrane) are found in the cytoplasmic fraction of osteoblasts. Overexpression of EGFP-tagged OA in osteoblasts showed that OA protein accumulates into vesicles for transportation to the cell membrane. We examined OA protein production in primary osteoblast cultures and found that OA is maximally expressed during the third week of culture (last stage of osteoblast differentiation). Glycosylation studies showed that OA isoform of 115 kDa is highly glycosylated. We also showed that retinoic acid (RA) stimulates the mannosylation of OA protein. In contrast, tunicamycin (TM) strongly inhibited N-glycans incorporation into OA protein. The functional role of the secreted OA isoform was revealed when cultures treated with anti-OA antibody, showed decreased osteoblast differentiation compared to untreated control cultures. Gain-of-function in osteoblasts using the pBABE viral system showed that OA overexpression in osteoblast stimulated their differentiation and function. The availability of a naturally occurring mutant mouse with a truncated OA protein provided further evidence that OA is an important factor for terminal osteoblast differentiation and mineralization. Using bone marrow mesenchymal cells derived from OA mutant and wild-type mice and testing their ability to differentiate into osteoblasts showed that differentiation of OA mutant osteoblasts was significantly reduced compared to wild-type osteoblasts. Collectively, our data suggest that OA acts as a positive regulator of osteoblastogenesis.

Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-β1 to induce mesenchymal cell condensation

Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor β1 (TGF‐β1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF‐β1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF‐β1 stimulation concomitant with dramatic up‐regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF‐β1‐induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up‐regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF‐β1 was also found to be mediated by CTGF. Immunofluorescence of developing mouse vertebrae showed that CTGF, TGF‐β1 and FN were co‐expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. Thus, CTGF is an essential downstream mediator of TGF‐β1‐induced MC condensation through its effects on cell proliferation and migration. CTGF is also involved in up‐regulating FN and suppressing Sox9 expression during TGF‐β1 induced MC condensation. J. Cell. Physiol. 210: 398–410, 2007.

Connective tissue growth factor (CTGF/CCN2) is a downstream mediator for TGF-beta1-induced extracellular matrix production in osteoblasts.

Connective tissue growth factor (CTGF/CCN2) is a cysteine‐rich, extracellular matrix (ECM) protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. Recent studies have identified CTGF as a downstream effector of transforming growth factor‐β1 (TGF‐β1) for certain functions in specific cell types. In this study, we examined the role of CTGF as a downstream mediator of TGF‐β1‐induced ECM production and cell growth in osteoblasts. Using primary cultures, we demonstrated that TGF‐β1 is a potent inducer of CTGF expression in osteoblasts, and that this induction occurred at all stages of osteoblast differentiation from the proliferative through mineralization stages. TGF‐β1 treatment of osteoblasts increased the expression and synthesis of the ECM components, collagen and fibronectin. When CTGF‐specific siRNA was used to prevent TGF‐β1 induction of CTGF expression, it also inhibited collagen and fibronectin production, thereby demonstrating the requirement of CTGF for their up‐regulation. To examine the effects of TGF‐β1 on osteoblast cell growth, cultures were treated with TGF‐β1 during the proliferative stage. Cell number was significantly reduced and the cells exhibited a decrease in G1 cyclin expression, consistent with TGF‐β1‐induced cell‐cycle arrest. Cultures transfected with CTGF siRNA prior to TGF‐β1 treatment showed an even greater reduction in cell number, suggesting that TGF‐β1‐induced growth arrest is independent of CTGF in osteoblasts. Collectively, these data demonstrate for the first time that CTGF is an essential downstream mediator for TGF‐β1‐induced ECM production in osteoblasts, but these two growth factors function independently regarding their opposing effects on osteoblast proliferation. J. Cell. Physiol. 210: 843–852, 2007.

Clopidogrel, a P2Y12 receptor antagonist, potentiates the inflammatory response in a rat model of peptidoglycan polysaccharide-induced arthritis.

The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists, which is irreversibly antagonized by the active metabolite of clopidogrel, a widely used anti-thrombotic drug. In this study, we investigated whether reduction of platelet reactivity leads to reduced inflammatory responses using a rat model of erosive arthritis. We evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of rats: 1) untreated, 2) clopidogrel-treated, 3) PG-PS-induced, and 4) PG-PS-induced and clopidogrel-treated. There were significant differences between the PG-PS+clopidogrel group when compared to the PG-PS group including: increased joint diameter and clinical manifestations of inflammation, elevated plasma levels of pro-inflammatory cytokines (IL-1 beta, interferon (IFN) gamma, and IL-6), an elevated neutrophil blood count and an increased circulating platelet count. Plasma levels of IL-10 were significantly lower in the PG-PS+clopidogrel group compared to the PG-PS group. Plasma levels of platelet factor 4 (PF4) were elevated in both the PG-PS and the PG-PS+clopidogrel groups, however PF4 levels showed no difference upon clopidogrel treatment, suggesting that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. Histology indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in PG-PS-induced arthritis animals. In summary, animals treated with clopidogrel showed a pro-inflammatory effect in the PG-PS-induced arthritis animal model, which might not be mediated by platelets. Elucidation of the mechanism of clopidogrel-induced cell responses is important to understand the role of the P2Y12 receptor in inflammation.

Performance of Repetitive Tasks Induces Decreased Grip Strength and Increased Fibrogenic Proteins in Skeletal Muscle: Role of Force and Inflammation

Background This study elucidates exposure-response relationships between performance of repetitive tasks, grip strength declines, and fibrogenic-related protein changes in muscles, and their link to inflammation. Specifically, we examined forearm flexor digitorum muscles for changes in connective tissue growth factor (CTGF; a matrix protein associated with fibrosis), collagen type I (Col1; a matrix component), and transforming growth factor beta 1 (TGFB1; an upstream modulator of CTGF and collagen), in rats performing one of two repetitive tasks, with or without anti-inflammatory drugs. Methodology/Results To examine the roles of force versus repetition, rats performed either a high repetition negligible force food retrieval task (HRNF), or a high repetition high force handle-pulling task (HRHF), for up to 9 weeks, with results compared to trained only (TR-NF or TR-HF) and normal control rats. Grip strength declined with both tasks, with the greatest declines in 9-week HRHF rats. Quantitative PCR (qPCR) analyses of HRNF muscles showed increased expression of Col1 in weeks 3–9, and CTGF in weeks 6 and 9. Immunohistochemistry confirmed PCR results, and also showed greater increases of CTGF and collagen matrix in 9-week HRHF rats than 9-week HRNF rats. ELISA, and immunohistochemistry revealed greater increases of TGFB1 in TR-HF and 6-week HRHF, compared to 6-week HRNF rats. To examine the role of inflammation, results from 6-week HRHF rats were compared to rats receiving ibuprofen or anti-TNF-α treatment in HRHF weeks 4–6. Both treatments attenuated HRHF-induced increases in CTGF and fibrosis by 6 weeks of task performance. Ibuprofen attenuated TGFB1 increases and grip strength declines, matching our prior results with anti-TNFα. Conclusions/Significance Performance of highly repetitive tasks was associated with force-dependent declines in grip strength and increased fibrogenic-related proteins in flexor digitorum muscles. These changes were attenuated, at least short-term, by anti-inflammatory treatments.

LPS-induced systemic inflammation is more severe in P2Y12 null mice

Thienopyridines are a class of antiplatelet drugs that are metabolized in the liver to several metabolites, of which only one active metabolite can irreversibly antagonize the platelet P2Y12 receptor. Possible effects of these drugs and the role of activated platelets in inflammatory responses have also been investigated in a variety of animal models, demonstrating that thienopyridines could alter inflammation. However, it is not clear whether it is caused only by the P2Y12 antagonism or whether off‐target effects of other metabolites also intervene. To address this question, we investigated P2Y12 KO mice during a LPS‐induced model of systemic inflammation, and we treated these KO mice with a thienopyridine drug (clopidogrel). Contrary to the reported effects of clopidogrel, numbers of circulating WBCs and plasma levels of cytokines were increased in LPS‐exposed KO mice compared with WT in this inflammation model. Moreover, both spleen and bone marrow show an increase in cell content, suggesting a role for P2Y12 in regulation of bone marrow and spleen cellular composition. Finally, the injury was more severe in the lungs of KO mice compared with WT. Interestingly, clopidogrel treatments also exerted protective effects in KO mice, suggesting off‐target effects for this drug. In conclusion, the P2Y12 receptor plays an important role during LPS‐induced inflammation, and this signaling pathway may be involved in regulating cell content in spleen and bone marrow during LPS systemic inflammation. Furthermore, clopidogrel may have effects that are independent of P2Y12 receptor blockade.

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Calcium Mobilization and Thromboxane Generation in Platelets

Objective—Members of the protein kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. We investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. Methods and Results—A pan-PKC inhibitor, GF109203X, potentiated ADP-induced cPLA2 phosphorylation and thromboxane generation as well as ERK activation and intracellular calcium (Ca2+i) mobilization, 2 signaling molecules, upstream of cPLA2 activation. Thus, PKCs inhibit cPLA2 activation by inhibiting ERK and Ca2+i mobilization. Because the inhibitor of classic PKC isoforms, GO-6976, did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP-induced thromboxane generation, calcium mobilization, and ERK phosphorylation were potentiated in PKC&egr; null murine platelets compared with platelets from wild-type littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKC&egr; knockout and wild-type was similar, suggesting that PKC&egr; does not affect ADP-induced aggregation directly. PKC&egr; knockout mice exhibited shorter times to occlusion in an FeCl3-induced arterial injury model and shorter bleeding times in tail-bleeding experiments. Conclusion—We conclude that PKC&egr; negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis.

Amelioration of inflammation, angiogenesis and CTGF expression in an arthritis model by a TSP1-derived peptide treatment.

Objective: To evaluate the effect of a thrombospondin 1 (TSP1)‐derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. Methods: Erosive arthritis in Lewis rats was induced by peptidoglycan‐polysaccharide (PG‐PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1‐derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT‐PCR. Results: Histological data indicated that the TSP1‐derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1‐derived peptide treatment. Also, immunofluorescence and RT‐PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1‐derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1‐derived peptide treated animals. Moreover, macrophage/monocyte‐specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1‐derived peptide treated animals. Conclusion: Both inflammation and angiogenesis were decreased after TSP1‐derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues. J. Cell. Physiol. 211: 504–512, 2007.