SERPINA3 is a marker of cartilage differentiation and is essential for the expression of extracellular matrix genes during early chondrogenesis.

Serine proteinase inhibitors (serpins) are a family of structurally similar proteins which regulate many diverse biological processes from blood coagulation to extracellular matrix (ECM) remodelling. Chondrogenesis involves the condensation and differentiation of mesenchymal stem cells (MSCs) into chondrocytes which occurs during early development. Here, and for the first time, we demonstrate that one serpin, SERPINA3 (gene name SERPINA3, protein also known as alpha-1 antichymotrypsin), plays a critical role in chondrogenic differentiation. We observed that SERPINA3 expression was markedly induced at early time points during in vitro chondrogenesis. We examined the expression of SERPINA3 in human cartilage development, identifying significant enrichment of SERPINA3 in developing cartilage compared to total limb, which correlated with well-described markers of cartilage differentiation. When SERPINA3 was silenced using siRNA, cartilage pellets were smaller and contained lower proteoglycan as determined by dimethyl methylene blue assay (DMMB) and safranin-O staining. Consistent with this, RNA sequencing revealed significant downregulation of genes associated with cartilage ECM formation perturbing chondrogenesis. Conversely, SERPINA3 silencing had a negligible effect on the gene expression profile during osteogenesis suggesting the role of SERPINA3 is specific to chondrocyte differentiation. The global effect on cartilage formation led us to investigate the effect of SERPINA3 silencing on the master transcriptional regulator of chondrogenesis, SOX9. Indeed, we observed that SOX9 protein levels were markedly reduced at early time points suggesting a role for SERPINA3 in regulating SOX9 expression and activity. In summary, our data support a non-redundant role for SERPINA3 in enabling chondrogenesis via regulation of SOX9 levels.

Matrix metalloproteinase-13 is fully activated by neutrophil elastase and inactivates its serpin inhibitor, alpha-1 antitrypsin: Implications for osteoarthritis.

Matrix metalloproteinase-13 (MMP-13) is a uniquely important collagenase that promotes the irreversible destruction of cartilage collagen in osteoarthritis (OA). Collagenase activation is a key control point for cartilage breakdown to occur, yet our understanding of the proteinases involved in this process is limited. Neutrophil elastase (NE) is a well-described proteoglycan-degrading enzyme which is historically associated with inflammatory arthritis, but more recent evidence suggests a potential role in OA. In this study, we investigated the effect of neutrophil elastase on OA cartilage collagen destruction and collagenase activation. Neutrophil elastase induced significant collagen destruction from human OA cartilage ex vivo, in an MMP-dependent manner. In vitro, neutrophil elastase directly and robustly activated pro-MMP-13, and N-terminal sequencing identified cleavage close to the cysteine switch at 72 MKKPR, ultimately resulting in the fully active form with the neo-N terminus of 85 YNVFP. Mole-per-mole, activation was more potent than by MMP-3, a classical collagenase activator. Elastase was detectable in human OA synovial fluid and OA synovia which displayed histologically graded evidence of synovitis. Bioinformatic analyses demonstrated that, compared with other tissues, control cartilage exhibited remarkably high transcript levels of the major elastase inhibitor, (AAT) alpha-1 antitrypsin (gene name SERPINA1), but these were reduced in OA. AAT was located predominantly in superficial cartilage zones, and staining enhanced in regions of cartilage damage. Finally, active MMP-13 specifically inactivated AAT by removal of the serine proteinase cleavage/inhibition site. Taken together, this study identifies elastase as a novel activator of pro-MMP-13 that has relevance for cartilage collagen destruction in OA patients with synovitis.

Evaluation of the primary stability in dental implants placed in low density bone with a new drilling technique, Osseodensification: an in vitro study.

BACKGROUND: Primary stability is an important key determinant of implant osseointegration. We investigated approaches to improve primary implant stability using a new drilling technique termed osseodensification (OD), which was compared with the conventional under-drilling (UD) method utilized for low-density bones. MATERIAL AND METHODS: We placed 55 conical internal connection implants in each group, in 30 low-density sections of pig tibia. The implants were placed using twist drill bits in both groups; groups Under Drilling (UD) and Osseodensification (OD) included bone sections subjected to conventional UD and OD drilling, respectively. Before placing the implants, we randomized the bone sections that were to receive these implants to avoid sample bias. We evaluated various primary stability parameters, such as implant insertion torque and resonance frequency analysis (RFA) measurements. RESULTS: The results showed that compared with implants placed using the UD technique, those placed using the OD technique were associated with significantly higher primary stability. The mean insertion torque of the implants was 8.87±6.17 Ncm in group 1 (UD) and 21.72±17.14 Ncm in group 2 (OD). The mean RFA was 65.16±7.45 ISQ in group 1 (UD) and 69.75±6.79 ISQ in group 2 (OD). CONCLUSIONS: The implant insertion torque and RFA values were significantly higher in OD group than in UD. Therefore, compared with UD, OD improves primary stability in low-density bones (based on torque and RFA measurements).

Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover.

The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices. The proteinase-activated receptor (PAR) family has a unique mechanism of activation requiring proteolysis of an extracellular domain forming a neo-N terminus that acts as a tethered ligand, a process that has been associated with the development of arthritis. Canonical PAR2 activation typically occurs via a serine proteinase at Arg36-Ser37, but other proteinases can cleave PARs downstream of the tethered ligand and "disarm" the receptor. To identify additional cleavage sites within PAR2, we synthesized a 42-amino-acid peptide corresponding to the extracellular region. We observed that all three soluble MMP collagenases, MMP-1, MMP-8, and MMP-13, cleave PAR2 and discovered a novel cleavage site (Ser37-Leu38). Metalloproteinases from resorbing bovine nasal cartilage and recombinant human collagenases could cleave a quenched fluorescent peptide mimicking the canonical PAR2 activation region, and kinetic constants were determined. In PAR2-overexpressing SW1353 chondrocytes, we demonstrated that the activator peptide SLIGKV-NH2 induces rapid calcium flux, inflammatory gene expression (including MMP1 and MMP13), and the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase. The corresponding MMP cleavage-derived peptide (LIGKVD-NH2) exhibited no canonical activation; however, we observed phosphorylation of ERK, providing evidence of biased agonism. Importantly, we demonstrated that preincubation with active MMP-1 reduced downstream PAR2 activation by a canonical activator, matriptase, but not SLIGKV-NH2 These results support a role for collagenases as proteinases capable of disarming PAR2, revealing a mechanism that suppresses PAR2-mediated inflammatory responses.

Cytokine-induced cysteine- serine-rich nuclear protein-1 (CSRNP1) selectively contributes to MMP1 expression in human chondrocytes.

Irreversible cartilage collagen breakdown by the collagenolytic matrix metalloproteinases (MMPs)-1 and MMP-13 represents a key event in pathologies associated with tissue destruction such as arthritis. Inflammation is closely associated with such pathology and occurs in both rheumatoid and osteoarthritis making it highly relevant to the prevailing tissue damage that characterises these diseases. The inflammation-induced activating protein-1 (AP-1) transcription factor is an important regulator of both MMP1 and MMP13 genes with interplay between signalling pathways contributing to their expression. Here, we have examined the regulation of MMP1 expression, and using in vivo chromatin immunoprecipitation analyses we have demonstrated that cFos bound to the AP-1 cis element within the proximal MMP1 promoter only when the gene was transcriptionally silent as previously observed for MMP13. Subsequent small interfering RNA-mediated silencing confirmed however, that cFos significantly contributes to MMP1 expression. In contrast, silencing of ATF3 (a prime MMP13 modulator) did not affect MMP1 expression whilst silencing of the Wnt-associated regulator cysteine- serine-rich nuclear protein-1 (CSRNP1) resulted in substantial repression of MMP1 but not MMP13. Furthermore, following an early transient peak in expression of CSRNP1 at the mRNA and protein levels similar to that seen for cFOS, CSRNP1 expression subsequently persisted unlike cFOS. Finally, DNA binding assays indicated that the binding of CSRNP1 to the AP-1 consensus-like sequences within the proximal promoter regions of MMP1 and MMP13 was preferentially selective for MMP1 whilst activating transcription factor 3 (ATF3) binding was exclusive to MMP13. These data further extend our understanding of the previously reported differential regulation of these MMP genes, and strongly indicate that although cFos modulates the expression of MMP1/13, downstream factors such as CSRNP1 and ATF3 ultimately serve as transcriptional regulators in the context of an inflammatory stimulus for these potent collagenolytic MMPs.

Protein kinase D3 modulates MMP1 and MMP13 expression in human chondrocytes.

Many catabolic stimuli, including interleukin-1 (IL-1) in combination with oncostatin M (OSM), promote cartilage breakdown via the induction of collagen-degrading collagenases such as matrix metalloproteinase 1 (MMP1) and MMP13 in human articular chondrocytes. Indeed, joint diseases with an inflammatory component are characterised by excessive extracellular matrix (ECM) catabolism. Importantly, protein kinase C (PKC) signalling has a primary role in cytokine-induced MMP1/13 expression, and is known to regulate cellular functions associated with pathologies involving ECM remodelling. At present, substrates downstream of PKC remain undefined. Herein, we show that both IL-1- and OSM-induced phosphorylation of protein kinase D (PKD) in human chondrocytes is strongly associated with signalling via the atypical PKCι isoform. Consequently, inhibiting PKD activation with a pan-PKD inhibitor significantly reduced the expression of MMP1/13. Specific gene silencing of the PKD isoforms revealed that only PKD3 (PRKD3) depletion mirrored the observed MMP repression, indicative of the pharmacological inhibitor specifically affecting only this isoform. PRKD3 silencing was also shown to reduce serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphorylation of all three mitogen-activated protein kinase groups. This altered signalling following PRKD3 silencing led to a significant reduction in the expression of the activator protein-1 (AP-1) genes FOS and JUN, critical for the induction of many MMPs including MMP1/13. Furthermore, the AP-1 factor activating transcription factor 3 (ATF3) was also reduced concomitant with the observed reduction in MMP13 expression. Taken together, we highlight an important role for PKD3 in the pro-inflammatory signalling that promotes cartilage destruction.

Differential effects of estrogen and raloxifene on messenger RNA and matrix metalloproteinase 2 activity in the rat uterus.

A detailed analysis of the differential effects of estrogen (E) compared to raloxifene (Ral), a selective estrogen receptor modulator (SERM), following estrogen receptor (ER) binding in gynecological tissues was conducted using gene microarrays, Northern blot analysis, and matrix metalloproteinase (MMP) 2 activity studies. We profiled gene expression in the uterus following acute (1 day) and prolonged daily (5 wk) treatment of E and Ral in ovariectomized rats. Estrogen regulated twice as many genes as Ral, largely those associated with catalysis and metabolism, whereas Ral induced genes associated with cell death and negative cell regulation. Follow-up studies confirmed that genes associated with matrix integrity were differentially regulated by Ral and E at various time points in uterine and vaginal tissues. Additional experiments were conducted to determine the levels of MMP2 activity in uterus explants from ovariectomized rats following 2 wk of treatment with E, Ral, or one of two additional SERMs: lasofoxifene, and levormeloxifene. Both E and lasofoxifene stimulated uterine MMP2 activity to a level twofold that of Ral, whereas levormeloxifene elevated MMP2 activity to a level 12-fold that of Ral. These data show that one of the significant differences between E and Ral signaling in the uterus is the regulation of genes and proteins associated with matrix integrity. This may be a potential key difference between the action of SERMs in the uterus of postmenopausal women.

Photochemical synthesis of N-arylbenzophenanthridine selective estrogen receptor modulators (serms).

Selective estrogen receptor modulators are an emerging class of pharmaceutically important molecules. Many compounds in this class contain an aminoethoxyaryl moiety attached to a polycyclic framework at an asymmetric carbon atom. To assess whether this carbon atom can be replaced by nitrogen, we have employed a Ninomiya enamide photocyclization for the rapid synthesis of a novel N-arylbenzophenanthridine framework, 4. Further elaboration of 4 into a new structural class of achiral, nonsteroidal estrogen receptor modulators is described.

LY353381.HCl: a novel raloxifene analog with improved SERM potency and efficacy in vivo.

Body weight, uteri, serum cholesterol and bones were shown previously in vivo to be sensitive to circulating levels of estrogen, as well as to synthetic, nonsteroidal ligands termed selective estrogen receptor modulators (SERM). In this study, we examined the in vivo effects of a new potent SERM on these tissues in 6-month-old, ovariectomized rats that were orally dosed with 0.0001-10 mg/kg/day LY353381.HCl for 5 weeks. LY353381.HCl prevented the ovariectomy-induced increase in body weight and serum cholesterol levels of treated rats and lowered them to below sham levels in a dose dependent manner, with maximum efficacy similar to estrogen or raloxifene. However, LY353381.HCl was consistently more potent than raloxifene, with a half maximal efficacious dose of 0.001 mg/kg for the reduction of body weight and cholesterol. In the uterus, LY353381.HCl had marginal effects on uterine weight compared to ovariectomized controls (OVX) like raloxifene, but unlike estrogen. Histological examination of uterine epithelial cell height showed little to no stimulatory effect of LY353381.HCl on the endometrium. Quantitative computed tomographic analyses (pQCT) of tibiae showed that LY353381.HCl prevented loss of bone due to ovariectomy with an ED50 of about 0.01 mg/kg with maximal efficacy observed at 0.1-1 mg/kg/day. Maximally attainable bone mineral density and content with LY353381.HCl were not significantly different from Sham or ovariectomized rats treated with estrogen or raloxifene. Interestingly, assessment of bone quality by biomechanical analyses showed that LY353381.HCl preserved the strength of the femora neck and midshaft, while improving the Young's modulus of cortical bone to beyond estrogen, raloxifene or sham levels. In uteri of immature rats treated with estrogen, LY353381.HCl antagonized the estrogen-induced elevation in uterine weight down to vehicle-dosed control levels with ED50 of 0.03 mg/kg/day. Therefore, LY353381.HCl was 30-100 times more potent than raloxifene in preventing ovariectomy effects on body weight, serum cholesterol and bone, while maintaining estrogen antagonist effects on the uterus. These animal data suggest that LY353381.HCl may have advantages over estrogen or raloxifene in the prevention of bone loss and treatment of other tissues in postmenopausal women.

Differential responses of estrogen target tissues in rats including bone to clomiphene, enclomiphene, and zuclomiphene.

The substituted triphenylethylene antiestrogen clomiphene (CLO) prevents cancellous bone loss in ovariectomized (OVX'd) rats. However, CLO is a mixture of two stereoisomers, enclomiphene (ENC) and zuclomiphene (ZUC), which have distinctly different activities on reproductive tissues and tumor cells. The purpose of the present dose response study was to determine the effects of ENC and ZUC on nonreproductive estrogen target tissues. These studies were performed in 7-month-old female rats with moderate cancellous osteopenia that was established by ovariectomizing rats 1 month before initiating treatment. OVX resulted in increases in body weight, serum cholesterol, endocortical resorption, and indices of cancellous bone turnover, as well as decreases in uterine weight, uterine epithelial cell height, bone mineral density, bone strength, and cancellous bone area. Estrogen treatment for 3 months restored body weight, uterine histology, dynamic bone measurements, and osteoblast and osteoclast surfaces in OVX'd rats to the levels found in the age-matched sham-operated rats. In contrast, estrogen only partially restored cancellous bone volume and uterine weight, and it reduced serum cholesterol to subnormal values. CLO was a weak estrogen agonist on uterine measurements and a much more potent agonist on body weight, serum cholesterol, and dynamic bone measurements. CLO increased trabecular thickness in osteopenic rats and was the most effective treatment in improving cancellous bone volume and architecture. ZUC was a potent estrogen agonist on all tissues investigated and had dose-dependent effects. In contrast, ENC had dose-dependent effects on most measurements similar to CLO and decreased the uterotrophic effects of ZUC. It is concluded that ENC antagonizes the estrogenic effects of ZUC on the uterus but that the beneficial effects of CLO on nonreproductive tissues in OVX'd rats is conferred by both isomers. Furthermore, the combined actions of the two isomers on bone volume and architecture were more beneficial than either isomer given alone.

Synthesis and pharmacology of conformationally restricted raloxifene analogues: highly potent selective estrogen receptor modulators.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator (SERM) which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. In vivo structure-activity relationships and molecular modeling studies have indicated that the orientation of the basic amine-containing side chain of 1, relative to the stilbene plane, is an important discriminating factor for the maintenance of tissue selectivity. We have constructed a series of analogues of 1 in which this side chain is held in an orientation which is orthogonal to the stilbene plane, similar to the low-energy conformation predicted for raloxifene. Herein, we report on the synthesis of these compounds and on their activity in a series of in vitro and in vivo biological assays reflective of the SERM profile. In particular, we describe their ability to (1) bind the estrogen receptor, (2) antagonize estrogen-stimulated proliferation of MCF-7 cells in vitro, (3) stimulate TGF-beta3 gene expression in cell culture, (4) inhibit the uterine effects of ethynyl estradiol in immature rats, and (5) potently reduce serum cholesterol and protect against osteopenia in ovariectomized (OVX) rats without estrogen-like stimulation of uterine tissue. These data demonstrate that one of these compounds, LY357489,4, is among the most potent SERMs described to date with in vivo efficacy on bone and cholesterol metabolism in OVX rats at doses as low as 0.01 mg/kg/d.

Development of a scintillation proximity assay for high-throughput measurement of intact parathyroid hormone.

A simple, high-throughput scintillation proximity assay (SPA) for parathyroid hormone (1-84) (PTH) has been developed. Fifteen commercially available N-terminal and six C-terminal anti-PTH antibodies were evaluated for detection of human PTH(1-84). Two C-terminal antibodies (CR1073M and 10-P55) gave the most consistent results. Using one of these antibodies (10-P55), an assay was developed with a sensitivity of 4 pg/ml for human and rat PTH(1-84). Porcine PTH(1-84) was not detectable. The intra-assay and inter-assay coefficients of variation for a 467 pg/ml sample were 6. 1 and 6.5%, respectively, and for a 21 pg/ml sample, 6.2 and 4.4%. Human PTH(1-34), while not detected in the assay, interfered with the detection of PTH(1-84). Smaller fragments [for example, human PTH(3-34)] and a C-terminal PTH fragment [PTH(53-84)] did not interfere in the assay. The procedure gave 106-110% recovery of human PTH(1-84) spiked into samples. Immunoreactive PTH concentrations in serum of rats administered EGTA were determined by SPA and by a commercially available PTH immunoassay. There was a good correlation between the two assays with significant increases in serum immunoreactive PTH concentrations at 15 and 30 min after EGTA injection and a rapid decrease to baseline values by 60 min. The SPA gives a high-throughput method for simply and accurately determining PTH(1-84) concentrations in serum.

Structure-activity relationships of selective estrogen receptor modulators: modifications to the 2-arylbenzothiophene core of raloxifene.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. A series of raloxifene analogs which contain modifications to the 2-arylbenzothiophene core have been prepared and evaluated for the ability to bind to the estrogen receptor and inhibit MCF-7 breast cancer cell proliferation in vitro. Their ability to function as tissue-selective estrogen agonists in vivo has been assayed in a short-term, ovariectomized (OVX) rat model with end points of serum cholesterol lowering, uterine weight gain, and uterine eosinophil peroxidase activity. These studies have demonstrated that (1) the 6-hydroxy and, to a lesser extent, the 4'-hydroxy substituents of raloxifene are important for receptor binding and in vitro activity, (2) small, highly electronegative 4'-substituents such as hydroxy, fluoro, and chloro are preferred both in vitro and in vivo, (3) increased steric bulk at the 4'-position leads to increased uterine stimulation in vivo, and (4) additional substitution of the 2-aryl moiety is tolerated while additional substitution at the 4-, 5-, or 7-position of the benzothiophene results in reduced biological activity. In addition, compounds in which the 2-aryl group is replaced by alkyl, cycloalkyl, and naphthyl substituents maintain a profile of in vitro and in vivo biological activity qualitatively similar to that of raloxifene. Several novel structural variants including 2-cyclohexyl, 2-naphthyl, and 6-carbomethoxy analogs also demonstrated efficacy in preventing bone loss in a chronic OVX rat model of postmenopausal osteopenia, at doses of 0.1-10 mg/kg.