Role played by serum, a biological cue, in the adherence of Enterococcus faecalis to extracellular matrix proteins, collagen, fibrinogen, and fibronectin.

BACKGROUND: Most previous studies have found that Enterococcus faecalis isolates do not show significant adherence to fibronectin and fibrinogen. METHODS: The influence of various conditions on E. faecalis adherence to extracellular matrix (ECM) proteins was evaluated using a radiolabeled-cell adherence assay. RESULTS: Among the conditions studied, growth in 40% horse serum (a biological cue with potential clinical relevance) elicited adherence of all 46 E. faecalis strains tested to fibronectin and fibrinogen but not to elastin; adherence levels were independent of strain source, and adherence was eliminated by treating cells with trypsin. As previously reported, serum also elicited adherence to collagen. Although prolonged exposure to serum during growth was needed for enhancement of adherence to fibrinogen, brief exposure (<5 >min) to serum had an immediate, although partial, enhancing effect on adherence to fibronectin and, to a lesser extent, collagen; pretreatment of bacteria with chloramphenicol did not decrease this enhanced adherence to fibronectin and collagen, indicating that protein synthesis is not required for the latter effect. CONCLUSION: Taken together, these data suggest that serum components may serve (1) as host environmental stimuli to induce the production of ECM protein-binding adhesin(s), as previously seen with collagen adherence, and also (2) as activators of adherence, perhaps by forming bridges between ECM proteins and adhesins.

Interactions and structural analysis of the zinc -binding motif in decorin, a small leucine rich proteoglycan in extracellular matrix

Decorin, a dermatan/chondroitin sulfate proteoglycan, is ubiquitously distributed in the extracellular matrix (ECM) of mammals. Decorin belongs to the small leucine rich proteoglycan (SLRP) family, a proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. The decorin core protein appears to mediate the binding of decorin to ECM molecules, such as collagens and fibronectin. It is believed that the interactions of decorin with these ECM molecules contribute to the regulation of ECM assembly, cell adhesions, and cell proliferation. These basic biological processes play critical roles during embryonic development and wound healing and are altered in pathological conditions such as fibrosis and tumorgenesis. ^ In this dissertation, we discover that decorin core protein can bind to Zn2+ ions with high affinity. Zinc is an essential trace element in mammals. Zn2+ ions play a catalytic role in the activation of many enzymes and a structural role in the stabilization of protein conformation. By examining purified recombinant decorin and its core protein fragments for Zn2+ binding activity using Zn2+-chelating column chromatography and Zn2+-equilibrium dialysis approaches, we have located the Zn2+ binding domain to the N-terminal sequence of the decorin core protein. The decorin N-terminal domain appears to contain two Zn2+ binding sites with similar high binding affinity. The sequence of the decorin N-terminal domain does not resemble any other reported zinc-binding motifs and, therefore, represents a novel Zn 2+ binding motif. By investigating the influence of Zn2+ ions on decorin binding interactions, we found a novel Zn2+ dependent interaction with fibrinogen, the major plasma protein in blood clots. Furthermore, a recombinant peptide (MD4) consisting of a 41 amino acid sequence of mouse decorin N-terminal domain can prolong thrombin induced fibrinogen/fibrin clot formation. This suggests that in the presence of Zn2+ the decorin N-terminal domain has an anticoagulation activity. The changed Zn2+-binding activities of the truncated MD4 peptides and site-directed mutagenesis generated mutant peptides revealed that the functional MD4 peptide might contain both a structural zinc-binding site in the cysteine cluster region and a catalytic zinc site that could be created by the flanking sequences of the cysteine cluster region. A model of a loop-like structure for MD4 peptide is proposed. ^

Characterization of dermatan sulfate proteoglycan 3 (DSPG3) and cartilage oligomeric matrix protein (COMP)

This dissertation describes the identification and characterization of human dermatan sulfate proteoglycan 3 (DSPG3) and the characterization of the transcriptional regulation of human cartilage oligomeric matrix protein (COMP) in cartilage, ligament, and tendon cells. DSPG3 and COMP are two extracellular matrix proteins. The function of these ECM proteins is unknown.^ DSPG3 was cloned, sequenced, and shown to be expressed in cartilage, ligament, and placenta. DSPG3 was mapped to human chromosome 12q21, and the genomic structure was identified. 1.6 kb of the promoter region has been sequenced, and several putative SOX9 sites were identified as well as 3 TATA sites. Furthermore, an evolutionary tree of the SLRP gene family, which includes DSPG3, is presented.^ The promoter region of COMP was cloned and sequenced. Several putative transcription factor binding sites were identified including multiple AP2 and SP1 sites. Three transcription start sites were found to be located directly downstream of one of the SP1 sites. In addition, the expression of COMP was demonstrated to be higher in tendon than in cartilage and ligament by both Northern and Western blot analysis, and several regions of the COMP promoter were shown to contain cell-specific regulatory elements. Analysis of the proximal 370bp region of the COMP promoter has also identified distinct patterns of nuclear protein binding for the three tissues, and two SP1 sites may play a role in the tissue-specific expression of COMP. ^

Skeletal abnormalities in mice lacking extracellular matrix proteins, thrombospondin-1, thrombospondin-3, thrombospondin-5, and type IX collagen.

Thrombospondin-5 (TSP5) is a large extracellular matrix glycoprotein found in musculoskeletal tissues. TSP5 mutations cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia; both show a characteristic growth plate phenotype with retention of TSP5, type IX collagen (Col9), and matrillin-3 in the rough endoplasmic reticulum. Whereas most studies focus on defining the disease process, few functional studies have been performed. TSP5 knockout mice have no obvious skeletal abnormalities, suggesting that TSP5 is not essential in the growth plate and/or that other TSPs may compensate. In contrast, Col9 knockout mice have diminished matrillin-3 levels in the extracellular matrix and early-onset osteoarthritis. To define the roles of TSP1, TSP3, TSP5, and Col9 in the growth plate, all knockout and combinatorial strains were analyzed using histomorphometric techniques. While significant alterations in growth plate organization were found in certain single knockout mouse strains, skeletal growth was only mildly disturbed. In contrast, dramatic changes in growth plate organization in TSP3/5/Col9 knockout mice resulted in a 20% reduction in limb length, corresponding to similar short stature in humans. These studies show that type IX collagen may regulate growth plate width; TSP3, TSP5, and Col9 appear to contribute to growth plate organization; and TSP1 may help define the timing of growth plate closure when other extracellular proteins are absent.

RNAi reduces expression and intracellular retention of mutant cartilage oligomeric matrix protein.

Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

Ribozyme-mediated reduction of wild-type and mutant cartilage oligomeric matrix protein (COMP) mRNA and protein.

Dominant-negative mutations in the homopentameric extracellular matrix glycoprotein cartilage oligomeric matrix protein (COMP) result in inappropriate intracellular retention of misfolded COMP in the rough endoplasmic reticulum of chondrocytes, causing chondrocyte cell death, which leads to two skeletal dysplasias: pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). COMP null mice show no adverse effects on normal bone development and growth, suggesting a possible therapy involving removal of COMP mRNA. The goal of this study was to assess the ability of a hammerhead ribozyme (Ribo56, designed against the D469del mutation) to reduce COMP mRNA expression. In COS7 cells transfected with plasmids that overexpress wild-type or mutant COMP mRNA and Ribo56, the ribozyme reduced overexpressed normal COMP mRNA by 46% and mutant COMP mRNA by 56% in a dose-dependent manner. Surprisingly, the use of recombinant adenoviruses to deliver wild-type or mutant COMP mRNA and Ribo56 simultaneously into COS7 cells proved problematic for the activity of the ribozyme to reduce COMP expression. However, in normal human costochondral cells (hCCCs) infected only with adenoviruses expressing Ribo56, expression of endogenous wild-type COMP mRNA was reduced in a dose-dependent manner by 50%. In chondrocytes that contain heterozygous COMP mutations (D469del, G427E and D511Y) that cause PSACH, Ribo56 was more effective at reducing COMP mRNA (up to 70%). These results indicate that Ribo56 is effective at reducing mutant and wild-type COMP levels in cells and suggests a possible mode of therapy to reduce the mutant protein load.

Studies of pseudoachondroplasia chondrocytes and knockdown of the cartilage oligomeric matrix protein

Cartilage oligomeric matrix protein (COMP) is a large, homopentameric, extracellular matrix glycoprotein. Mutations in COMP cause two skeletal dysplasias: pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EMD1). These dwarfing conditions are caused by retention of misfolded mutant COMP with type IX collagen and matrilin-3 (MATN3) in the rough endoplasmic reticulum (rER) of the chondrocyte. These proteins form a matrix in the rER that continues to expand until it fills the entire cell, eventually causing cell death. Interestingly, loss of COMP in COMP null mice does not affect normal bone development or growth, suggesting that elimination of COMP (wildtype and mutant) expression may prevent PSACH. The hypothesis of these studies was that a hammerhead ribozyme could eliminate or knockdown COMP mRNA expression in PSACH chondrocytes . To test this hypothesis, a human chondrocyte model system that recapitulates the PSACH chondrocyte phenotype was developed by over-expressing mutant (mt-) COMP in normal chondrocytes using a recombinant adenovirus. Chondrocytes over-expressing mt-COMP developed giant rER cisternae containing COMP, type IX collagen and MATN3. Deconvolution microscopy and computer modeling showed that these proteins formed an ordered matrix surrounding a type II pro-collagen core. Additionally, the results show that a hammerhead ribozyme, ribozyme 56 (Ribo56) reduced over-expressed mt-COMP in COS cells and endogenous COMP in normal chondrocytes and mt-COMP in three PSACH chondrocytes cell line (with different mutations) by 40-70%. Altogether, these studies show that the PSACH cellular phenotype can be created in vitro and that the mt-COMP protein burden can be reduced by the presence of a COMP-specific ribozyme. Future studies will focus on designing ribozymes or short interfering RNA (siRNA) technologies that will result in better knockdown of COMP expression as well as the temporal constraints imposed by the PSACH phenotype. ^

Fibulin-2 stabilizes tumor extracellular matrix and drives malignant progression of lung adenocarcinoma

The ECM of epithelial carcinomas undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How tumors maintain ECM integrity in the face of dynamic biophysical forces is still largely unclear. This study addresses these deficiencies using mouse models of human lung adenocarcinoma. Spontaneous lung tumors were marked by disorganized basement membranes, dense collagen networks, and increased tissue stiffness. Metastasis-prone lung adenocarcinoma cells secreted fibulin-2 (Fbln2), a matrix glycoprotein involved in ECM supra-molecular assembly. Fibulin-2 depletion in tumor cells decreased the intra-tumoral abundance of matrix metalloproteinases and reduced collagen cross-linking and tumor compressive properties resulting in inhibited tumor growth and metastasis. Fbln2 deposition within intra-tumoral fibrotic bands was a predictor of poor clinical outcome in patients. Collectively, these findings support a feed-forward model in which tumor cells secrete matrix-stabilizing factors required for the assembly of ECM that preferentially favors malignant progression. To our knowledge, this is the first evidence that tumor cells directly regulate the integrity of their surrounding matrix through the secretion of matrix-stabilizing factors such as fibulin-2. These findings open a new avenue of research into matrix assembly molecules as potential therapeutic targets in cancer patients.

Adherence to host extracellular matrix and serum components by Enterococcus faecium isolates of diverse origin.

Enterococcus faecium has emerged as an important cause of nosocomial infections over the last two decades. We recently demonstrated collagen type I (CI) as a common adherence target for some E. faecium isolates and a significant correlation was found to exist between acm-mediated CI adherence and clinical origin. Here, we evaluated 60 diverse E. faecium isolates for their adherence to up to 15 immobilized host extracellular matrix and serum components. Adherence phenotypes were most commonly observed to fibronectin (Fn) (20% of the 60 isolates), fibrinogen (17%) and laminin (Ln) (13%), while only one or two of the isolates adhered to collagen type V (CV), transferrin or lactoferrin and none to the other host components tested. Adherence to Fn and Ln was almost exclusively restricted to clinical isolates, especially the endocarditis-enriched nosocomial genogroup clonal complex 17 (CC17). Thus, the ability to adhere to Fn and Ln, in addition to CI, may have contributed to the emergence and adaptation of E. faecium, in particular CC17, as a nosocomial pathogen.

A study of the biology of perlecan

Extracellular matrix (ECM) is a component of a variety of organisms that provides both structural support and influence upon the cells it surrounds. The importance of the ECM is becoming more apparent as matrix defects are linked to human disease. In this study, the large, extracellular matrix heparan sulfate proteoglycan, perlecan (Pln) is examined in two systems. First, the role of Pln in the interaction between a blastocyst and uterine epithelial cells is investigated. In mice, blastocyst attachment and implantation occurs at approximately d 4.5 post coitus. In addition, a delayed implantation model has been used to distinguish between the response of the blastocyst to that of hatching and of becoming attachment competent. ^ The second series of experiments described in this study focuses on the process of chondrogenesis in mice. Pln, commonly expressed with other basement membrane (BM) proteins, was found to be expressed in cartilaginous tissue without other BM proteins. This unusual expression pattern led to further study and the development of an in vitro chondrogenesis assay using the mouse embryonic fibroblast cell line, C3H/10T1/2. When cultured on Pln in vitro, these cells form aggregates and express the cartilage proteins, collagen type II and aggrecan. In examining the participation of the heparan sulfate (HS) chains in this process, the proteoglycan was enzymatically digested to remove the HS chains before the initiation of 10T1/2 cell culture. After digestion, the ability of Pln to stimulate aggregate formation was greatly diminished. Thus, the HS chains participate in the cell induction process. To determine which domain of Pln might be responsible for this activity, recombinant fragments of Pin were used in the cell culture assay. Of all recombinant protein fragments tested, only the domain including the HS chains, domain 1, was able to initiate the morphological change exhibited by the 10T1/2 cells. Similar to native Pln, when HS chains were removed from domain I, chondrogenic activity was abolished. A variant of domain I carrying both HS and chondroitin sulfate (CS) chains retained activity when only HS chains were removed. When both HS and CS chains were removed, then activity was lost. ^ The ability to rapidly stimulate differentiation of 10T1/2 cells in vitro may lead to better control of chondrogenesis in vitro and in vivo, providing better understanding and manipulation of the chondrogenic process. This greater understanding may have benefits for study of cartilage and bone diseases and subsequent treatment options. (Abstract shortened by UMI.)^

Osteopontin and cadherin 11 are novel mediators and drug targets for chronic lung diseases

Chronic lung diseases (CLDs) are a considerable source of morbidity and mortality and are thought to arise from dysregulation of normal wound healing processes. An aggressive, feature of many CLDs is pulmonary fibrosis (PF) and is characterized by excess deposition of extracellular matrix (ECM) proteins from myofibroblasts in airways. However, factors regulating myofibroblast biology are incompletely understood. Proteins in the cadherin family contribute epithelial to mesenchymal transition (EMT), a suggested source of myofibroblasts. Cadherin 11 (CDH11) contributes to developmental and pathologic processes that parallel those seen in PF and EMT. Utilizing Cdh11 knockout (Cdh11 -/-) mice, the goal of this study was to characterize the contribution of CDH11 in the bleomycin model of PF and assess the feasibility of treating established PF. We demonstrate CDH11 in macrophages and airway epithelial cells undergoing EMT in lungs of mice given bleomycin and patients with PF. Endpoints consistent with PF including ECM production and myofibroblast formation are reduced in CDH11-targeted mice given bleomycin. Findings suggesting mechanisms of CDH11-dependent fibrosis include the regulation of the profibrotic mediator TGF-â in alveolar macrophages and CDH11-mediated EMT. The results of this study propose CDH11 as a novel drug target for PF. In addition, another CLD, chronic obstructive pulmonary disease (COPD), is characterized by airway inflammation and destruction. Adenosine, a nucleoside signaling molecule generated in response to cell stress is upregulated in patients with COPD and is suggested to contribute to its pathogenesis. An established model of adenosine-mediated lung injury exhibiting features of COPD is the Ada -/- mouse. Previous studies in our lab suggest features of the Ada -/- phenotype may be secondary to adenosine-dependent expression of osteopontin (OPN). OPN is a protein implicated in a variety of human pathology, but its role in COPD has not been examined. To address this, Ada/Opn -/- mice were generated and endpoints consistent with COPD were examined in parallel with Ada -/- mice. Results demonstrate OPN-mediated pulmonary neutrophilia and airway destruction in Ada -/- mice. Furthermore, patients with COPD exhibit increased OPN in airways which correlate with clinical airway obstruction. These results suggest OPN represents a novel biomarker or therapeutic target for the management of patients with COPD. The importance of findings in this thesis is highlighted by the fact that no pharmacologic interventions have been shown to interfere with disease progression or improve survival rates in patients with COPD or PF.

Role of NonO-histone interaction in TNFalpha-suppressed prolyl-4-hydroxylase alpha1.

Inflammation is a key process in cardiovascular diseases. The extracellular matrix (ECM) of the vasculature is a major target of inflammatory cytokines, and TNFalpha regulates ECM metabolism by affecting collagen production. In this study, we have examined the pathways mediating TNFalpha-induced suppression of prolyl-4 hydroxylase alpha1 (P4Halpha1), the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Using human aortic smooth muscle cells, we found that TNFalpha activated the MKK4-JNK1 pathway, which induced histone (H) 4 lysine 12 acetylation within the TNFalpha response element in the P4Halpha1 promoter. The acetylated-H4 then recruited a transcription factor, NonO, which, in turn, recruited HDACs and induced H3 lysine 9 deacetylation, thereby inhibiting transcription of the P4Halpha1 promoter. Furthermore, we found that TNFalpha oxidized DJ-1, which may be essential for the NonO-P4Halpha1 interaction because treatment with gene specific siRNA to knockout DJ-1 eliminated the TNFalpha-induced NonO-P4Halpha1 interaction and its suppression. Our findings may be relevant to aortic aneurysm and dissection and the stability of the fibrous cap of atherosclerotic plaque in which collagen metabolism is important in arterial remodeling. Defining this cytokine-mediated regulatory pathway may provide novel molecular targets for therapeutic intervention in preventing plaque rupture and acute coronary occlusion.

A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.

The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (DeltaCbfa1) in differentiated osteoblasts only postnatally. DeltaCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. DeltaCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that DeltaCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally.

Molecular mechanisms of chondrocyte-specific expression on the mouse pro$\alpha$1(II) collagen gene

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro$\alpha1$(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a $\beta$-galactosidase reporter gene. A construction containing a 3000-bp promoter and a 3020-bp intron 1 fragment directed high levels of $\beta$-galactosidase expression specifically to chondrocytes. Successive deletions of intron 1 delineated a 48-bp fragment which targeted $\beta$-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. When the Col2a1 promoter was replaced with a minimal $\beta$-globin promoter, the 48-bp intron 1 sequence was still able to target expression of the transgene to chondrocytes, specifically. Therefore a 48-bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression to a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression. Nuclear proteins present selectively in mouse primary chondrocytes and rat chondrosarcoma cells bind to the three putative HMG (High-Mobility-Group) domain protein binding sites in this 48-bp sequence and the chondrocyte-specific proteins likely bind the DNA through minor groove. Together, my results indicate that a 48-bp sequence in Col2a1 intron 1 controls chondrocyte-specific expression in vivo and suggest that chondrocytes contain specific nuclear proteins involved in enhancer activity. ^

Collagen I modulates growth and gene expression in prostate cancer cells

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to skeletal tissue. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as extracellular matrix (ECM) components, have been implicated in the spread and propagation of prostatic carcinoma. In these studies, we have utilized the PC3 cell line, derived from a human bone metastasis, to investigate the influence of the predominant bone ECM protein, type I collagen, on prostate cancer cell proliferation and gene expression. We have also initiated the design and production of ribozymes to specific gene targets that may influence prostate cancer bone metastasis. ^ Our results demonstrate that PC3 cells rapidly adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-L-lysine (PLL). Flow cytometry analysis reveals the presence of the α1, α2 and α3 collagen binding integrin subunits. The use of antibody function blocking studies reveals that PC3 cells can utilize α2β 1 and α3β1 integrins to adhere to collagen I. Once plated on collagen I, the cells exhibit increased rates of proliferation compared with cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of proteins associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. To further characterize the effect of type I collagen on prostate cancer bone metastasis, we utilized a cDNA microarray strategy to monitor type I collagen-mediated changes in gene expression. Results of this analysis revealed a gene expression profile reflecting the increased proliferation occurring on type I collagen. Microarray analysis also revealed differences in the expression of specific gene targets that may impact on prostate cancer metastasis to bone. ^ As a result of our studies on the interaction of prostate cancer cells and the skeletal ECM, we sought to develop novel molecular tools for future gene therapy of functional knockdown experiments. To this end, we developed a series of ribozymes directed against the α2 integrin and at osteopontin, a protein implicated in the metastasis of various cancers, including prostate. These ribozymes should facilitate the future study of the mechanism of prostate cancer cell proliferation, and disease progression occurring at sites of skeletal metastasis where a type I collagen-based environment predominates. ^ Together these studies demonstrate the involvement of bone ECM proteins on prostate cancer cell proliferation and suggest that they may play a significant role on the growth of prostate metastases once in the bone microenvironment. ^