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Articular cartilage functions to reduce the mechanical stresses associated with diarthrodial joint movement, protecting these joints over a lifetime of use. Tissue function is maintained through the balance between synthesis and resorption (i.e., metabolism) of extracellular matrix (ECM) by articular chondrocytes (ACs). Two important hormonal regulators of cartilage metabolism are interleukin-1 (IL-1) and insulin-like growth factor-I (IGF-I). These factors have antagonistic effects on chondrocyte activity, and during the progression of osteoarthritis, IL-1 is thought to promote chondrocyte hyporesponsiveness to IGF-I. To better understand how the anabolic (IGF-I) and catabolic (IL-1) stimuli are linked within articular cartilage, we examined the mechanisms by which IL-1 regulates the IGF-I signaling system of ACs. Equine chondrocytes from non-arthritic stifle joints were multiplied over serial passages, re-differentiated in alginate beads, and stimulated with recombinant equine IL-1β. Chondrocytes were assayed for type I IGF receptor (IGF-IR), IGF binding proteins (IGFBPs), and endogenously-secreted IGF-I. Our experimental findings solidify the significance of IL-1 as a key regulator of IGF-I signaling within articular cartilage, demonstrating that regulation of the IGF-I system occurs through both direct (transcription) and indirect (proteolysis) mechanisms. These results have implications for molecular therapies (e.g., gene transfer) directed at reversing osteoarthritic cartilage deterioration. The presented research concerns not only cartilage biology but also tissue engineering strategies for cartilage repair. Alginate hydrogel culture has been reported to re-establish chondrocytic phenotype following monolayer expansion, but studies have not addressed effects on the signaling systems responsible for chondrocyte metabolism. We investigated whether chondrocyte culture history influences the IGF-I system and its regulation by IL-1. ACs expanded by serial passaging were either encapsulated in alginate beads or maintained on tissue culture plastic (TCP). Bead and TCP cells were plated at high-density, stimulated with IL-1β, and assayed for expression of IGF-I signaling mediators. Intermediate alginate culture yielded disparate basal levels of IGF-IR and IGFBP-2, which were attributed to differential transcription. The distinct mediator profiles coincided with varied effects of exogenous IL-1β and IGF-I on collagen Ia1 expression and cell growth rate. This study demonstrates that culture strategy impacts the IGF-I system of ACs, likely impacting their capability to mediate cartilage repair. / Ph. D.

The overall objective of this research was to understand the mechanims by which growth hormone (GH) regulates insulin-like growth factor-I (IGF-I) gene expression in liver. Previous studies have suggested that GH regulation of IGF-I gene expression in liver is mediated by binding of the transcription factor signal transducer and activator of transcription (STAT) 5 to four binding sites located distantly from the IGF-I promoter. The first specific objective of this research was to determine whether additional STAT5 binding sites were involved in GH stimulation of IGF-I gene expression in liver. Sequence analysis of 170 kb of mouse genomic DNA revealed nineteen consensus STAT5 binding sequences corresponding to fourteen ~200 bp chromosomal regions that were conserved in the corresponding human DNA sequence. Eight of these chromosomal regions were able to mediate STAT5 activation of reporter gene expression in cotransfection experiments. Two of these chromosomal regions corresponded to those previously identified. Gel-shift assays indicated that the eight new STAT5 binding sites and three of the four previously identified STAT5 binding sites could bind GH-activated STAT5 from mouse liver. Together, these results suggest that GH stimulation of IGF-I gene transcription in the mouse liver may be mediated by at least eleven STAT5 binding sites located distantly from the IGF-I promoter. In a previous study, I found that liver expression of liver-enriched transcription factor hepatocyte nuclear factor 3γ (HNF-3γ) was increased by GH in cattle. Therefore, the second specific objective of this research was to determine how GH stimulates HNF-3γ gene expression and whether the increased HNF-3γ mediates GH stimulation of IGF-I gene expression in bovine liver. Sequence analysis of the bovine HNF-3γ promoter revealed the presence of two putative binding sites for STAT5. The proximal putative STAT5 binding site appears to be conserved in other mammals. Chromatin immunoprecipitation (ChIP) assays demonstrated that GH increased the binding of STAT5 to the HNF-3γ promoter in bovine liver and that this binding was associated with increased HNF-3γ expression. Gel-shift assays demonstrated that the proximal STAT5 binding site in the HNF-3γ promoter could bind GH-activated STAT5 from bovine liver. Cotransfection analyses showed that the proximal STAT5 binding site was necessary for the HNF-3γ promoter to be activated by GH. The promoter of the bovine IGF-I gene contains three putative HNF-3 binding sites that seem to be evolutionarily conserved. ChIP assays indicated that GH stimulated the binding of HNF-3γ to the IGF-I promoter in bovine liver. Gel-shift assays showed that one of the putative HNF-3 binding sites could bind HNF-3γ protein from bovine liver. Co-transfection analyses demonstrated that this HNF-3 binding site was necessary for HNF-3γ activation of reporter gene expression from the IGF-I promoter. In summary, the results of this dissertation research suggest that GH-activated STAT5 directly stimulates IGF-I gene transcription in liver by binding to at least eleven distantly located STAT5 binding sites in the IGF-I locus and indirectly stimulates IGF-I gene transcription by enhancing HNF-3γ gene expression in the liver. / Ph. D.

Obesity is a worldwide epidemic increasing at an alarming rate among youth who are facing similar health problems as adults. Sleep Apnea Syndrome (SAS) is an underdiagnosed comorbidity of obesity, characterized by repetitive nocturnal interruptions in breathing. Obesity is associated with delayed skeletal maturation in overweight youth, but mechanisms contributing to this problem are unclear. Obesity and SAS both have been shown to disrupt regulatory hormones and cytokines that influence bone accretion during adolescence. PURPOSE: The purpose of this study was to assess the combined effects of excess body weight and SAS on bone mineral density (BMD) and content (BMC), bone turnover, and on the regulatory hormones leptin and IGF-1 known to potentially influence bone accretion during adolescence. METHODS: Men aged 18-28 years were assigned to groups as follows: normal weight controls (CON: AHI <3, n=8); overweight without SAS (OWT: BMI < 26 kg/m2 and AHI <3, n=9); and overweight with SAS (SAS: BMI >26 kg/m2 and AHI >5, n=8). The apnea/hypopnea index (AHI) expresses the score for disrupted nighttime breathing events/hr and was obtained in this study with results from a home sleep screening test. Health history and Epworth Sleepiness Scale (ESS) questionnaires also were administered. Bone mineral parameters and body composition variables were measured with dual-energy X-ray absorptiometry. Serum osteocalcin, leptin, IGF-1, and NTx-1 were measured, respectively, by radioimmunoassay and enzyme-linked immunoabsorbent assay. RESULTS: Fat-free mass, intra-abdominal fat, and fat mass were higher in the SAS and OWT groups (p<0.03). ESS scores revealed that SAS individuals were sleepier than CON and OWT groups (p<0.009). Total body and site-specific BMD and BMC values (lumbar spine, hip, and forearm) were similar between groups and did not relate to the estimated AHI score. Serum OC and NTx-1 did not differ between groups. Leptin levels were 30% higher in OWT and SAS than in the CON group (p<0.02), but did not correlate with the AHI score. Across all subjects (n=25), only lumbar spine BMC (p<0.005) was correlated to AHI (r=-.52; p<0.01). The preponderance of this relationship between AHI and lumbar spine BMC was attributable to the close inverse association of these two variables within the SAS group (r = -.81; p<0.001). CONCLUSION: The effects of SAS were not influenced by the amount of whole-body, intra-abdominal adiposity or lean body mass. Neither leptin nor IGF-1 predicted bone status across all groups. Daytime fatigue and sleepiness, a cardinal symptom of SAS, combined with overweight may contribute to lower lumbar BMC by chronically reducing weight-bearing physical activity and thereby reduce exposure time for mechanical loading of the spine in affected individuals. Further research is needed to explore the biochemical, physiological, and apparently the physical activity implications of SAS on skeletal status and turnover. / Master of Science