Human osteoarthritis is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multifactorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecan is cleaved at a specific 'aggrecanase' site in human osteoarthritic cartilage; this cleavage can be performed by several members of ADAMTS family of metalloproteases. The relative contribution of individual ADAMTS proteases to cartilage destruction during osteoarthritis has not been resolved. Here we describe experiments with a genetically modified mouse in which the catalytic domain of ADAMTS5 (aggrecanase-2) was deleted. After surgically induced joint instability, there was significant reduction in the severity of cartilage destruction in the ADAMTS5 knockout mice compared with wild-type mice. This is the first report of a single gene deletion capable of abrogating the course of cartilage destruction in an animal model of osteoarthritis. These results demonstrate that ADAMTS5 is the primary 'aggrecanase' responsible for aggrecan degradation in a murine model of osteoarthritis, and suggest rational strategies for therapeutic intervention in osteoarthritis.

Objective. To determine the importance of the enzymatic activity of ADAMTS-4 in normal growth and development and to evaluate the role of ADAMTS-4 in the progression of osteoarthritis (OA).Methods. We generated catalytic domain-deleted ADAMTS-4-transgenic mice and performed extensive gross and histologic analyses of various organs. The mice were challenged by surgical induction of joint instability leading to OA, to determine the importance of the enzymatic activity of ADAMTS-4 in the progression of the disease. The response of wild-type (WT) and ADAMTS-4-knockout (ADAMTS-4-KO) articular cartilage to interleukin-1 and retinoic acid challenge in vitro was also evaluated.Results. ADAMTS-4-KO mice up to 1 year of age exhibited no gross or histologic abnormalities in 36 tissue sites examined. Despite evidence of ADAMTS-4 expression and activity in growth plates of WT mice, catalytic silencing of this proteinase caused no abnormalities in skeletal development, growth, or remodeling. There was no effect of ADAMTS-4 knockout on the progression or severity of OA 4 weeks or 8 weeks after surgical induction of joint instability. Enzymatic cleavage of aggrecan at the TEGE 373-374 ARGS site was clearly evident after exposure of articular cartilage from ADAMTS-4-KO mice to inflammatory cytokines. Conclusion. Although expression of the ADAMTS-4 gene has been found in many tissuesthroughout the body, deletion of enzymatic activity did not appear to have any effect on normal growth and physiology. Our study provides evidence that ADAMTS-4 is the primary aggrecanase in murine growth plates; however, deletion of its enzymatic activity did not affect normal long bone remodeling. Our results also lead to the hypothesis that, in the mouse, ADAMTS-4 is not the primary enzyme responsible for aggrecan degradation at the TEGE 373-374 ARGS site. The elucidation of the relative importance of ADAMTS-4 in the pathologic process of human OA will require examination of human OA tissues and evidence of disease modification in patients following therapeutic intervention.

Objective. To phenotypically characterize ADAMTS-4-and ADAMTS-5-double-knockout mice, and to determine the effect of deletion of ADAMTS-4 and ADAMTS-5 on the progression of osteoarthritis (OA) in mice.Methods. Mice lacking the catalytic domain of ADAMTS-4 and ADAMTS-5 were crossed to generate ADAMTS-4/5-double-knockout animals. Twelve-weekold and 1-year-old male and female ADAMTS-4/5-double-knockout mice were compared with age-and sex-matched wild-type (WT) mice by evaluating terminal body weights, organ weights, clinical pathology parameters, PIXImus mouse densitometry findings, and macroscopic and microscopic observations. ADAMTS-4/5-double-knockout mice were challenged by surgical induction of joint instability to determine the importance of these genes in the progression of OA. Articular and nonarticular cartilage explants from WT and ADAMTS-4/5-double-knockout mice were treated with interleukin-1 (IL-1) plus retinoic acid ex vivo, to examine proteoglycan degradation.Results. There were no genotype-related phenotype differences between ADAMTS-4/5-double-knockout and WT mice through 1 year of age, with the exception that female ADAMTS-4/5-double-knockout mice had a lower mean terminal body weight at the 12-week time point. Eight weeks after surgical induction of joint instability, OA was significantly less severe in ADAMTS-4/5-doubleknockout mice compared with WT mice. Following stimulation of cartilage explants with IL-1 plus retinoic acid, aggrecanase-mediated degradation in ADAMTS-4/5-double-knockout mice was ablated, to a level comparable with that in ADAMTS-5-knockout mice.Conclusion. Dual deletion of ADAMTS-4 and ADAMTS-5 generated mice that were phenotypically indistinguishable from WT mice. Deletion of ADAMTS-4/5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine OA. These effects in the ADAMTS-4/5-doubleknockout mice were comparable with those observed with deletion of ADAMTS-5 alone.

Gob-5 is a member of the calcium-activated chloride channel family and has been associated with allergic response in mouse models of pulmonary inflammation. Gene expression of Gob-5 has been shown to be induced in allergic airways and has been strongly associated with mucin gene regulation and goblet cell hyperplasia. We investigated the physiologic role of Gob-5 in murine models of pulmonary inflammation using mice deficient in Gob-5. After sensitization and aerosol challenge with ovalbumin (OVA), Gob-5 knockout mice exhibit significantly increased bronchoalveolar lavage (BAL) inflammation as compared with wild-type controls. The augmented inflammation in BAL consisted predominantly of neutrophils. Examination of perivascular inflammation revealed that tissue inflammation was decreased in OVA-challenged Gob-5-/- mice. OVA-challenged Gob-5 knockout mice also had decreased goblet cell hyperplasia as well as decreased mucus production. These mice also had decreased airway hypersensitivity after cholinergic provocation with methacholine. Gob-5 knockout mice were also challenged via intranasal LPS, a TLR-4 agonist. Gob-5-/- mice responded with increased neutrophilic BAL inflammation and decreased perivascular tissue inflammation as compared with wild-type controls. There was little effect on goblet cell hyperplasia and mucus production after LPS challenge. These observations reinforce findings that associate Gob-5 with goblet cell hyperplasia and mucus production in the allergic immune response, but also implicate Gob-5 in the regulation of tissue inflammation in the innate immune response.

Neurons in the hypothalamus of estrogen receptor alpha-knockout (ER alphaKO) mice have been shown to concentrate radiolabeled estrogen and estrogen treatment regulates the expression of progesterone receptor mRNA. The purpose of the present study was to utilize in situ hybridization histochemistry to determine the anatomical distribution of ER beta mRNA in ER alphaKO mouse forebrain. The results of these studies revealed an extensive distribution of ER beta mRNA in the hypothalamic regions including medial preoptic area, suprachiasmatic nucleus, paraventricular nucleus, dorsomedial nucleus, medial tuberal nucleus, and the premammillary nuclei. Additional labeled perikarya were also detected in the glomerular layer of the olfactory bulb; tenia tecta; anterior septum; bed nucleus of the stria terminalis; medial, basolateral and cortical nuclei of the amygdala; cerebral and entorhinal cortex; the septohippocampal nucleus; Ammon's horn of the hippocampus and the dorsal raphe. The results of these in situ hybridization histochemical studies have provided novel information about the distribution of ER beta mRNA in the ER alphaKO mouse forebrain. In addition, these morphological data provides evidence that estrogen may exert its actions in the ER alphaKO mouse brain via ER beta and thereby maintain organizational and activational effects.