Probes for KIT

Control of seasonal reproduction in horses has yet to be fully elucidated; however, duration of melatonin secretion is a seasonal regulator of the hypothalamic-pituitary-gonadal (HPG) axis. The hypophyseal pars tuberalis (PT) appears to play a key role in the transduction of melatonin signal through melatonin responsive, PT-specific cells that produce thyroid stimulating hormone (TSH). The objectives of these experiments were to characterize and colocalize TSH-producing cells and melatonin receptors (MT1r) in the PT during breeding and non-breeding seasons. Pituitaries from nine light-horse type mares, aged 6 to 26 years, were obtained following euthanasia during the breeding and non-breeding seasons. Mares were determined to be either cycling (n = 5) or seasonally anovulatory (n = 4) based on season, gross inspection of ovarian structures, and plasma progesterone concentrations. Immediately following euthanasia, the carotid artery was perfused with sodium nitrite and 4% paraformaldehyde before decapitation and dissection of PT from the hypophysis. Immunofluorescent detection of TSH was carried out on fixed-frozen PT sections and number of immunopositive cells per 1 mm2 were counted and averaged for three replications per horse to compare TSH distribution between breeding and non-breeding season using Mann Whitney U test. TSH-immunoreactive cells were present in PT and were greater (P < 0.05) in PT during the breeding season comparedto non-breeding season; few to no immunoreactive cells were seen in PT from non-breeding season. In situ hybridization (ISH) was carried out using RNAscope HD Red Detection Kit and a custom MT1r probe (Ec-MTNR1A) to confirm MT1r localization and compare MT1r distribution between breeding and non-breeding seasons. Mean percentage of MT1r immunopositive cells, determined as a percentage of total number of cells, and expression of MT1r mRNA per cell, determined as mean number of dots (representing mRNA) per cell, were analyzed using Mann Whitney U test. Melatonin receptor mRNA was densely expressed in glandular cells of the PT and no differences were found in either percent immunopositive cells or mean number of mRNA dots per cell between breeding and non-breeding seasons. To determine if MT1r colocalize with TSH-producing cells, ISH was carried out alongside immunofluorescent detection of TSH as previously described. In cycling mares, MT1r mRNA colocalized with TSH-producing cells in the PT; however, not all TSH-producing cells colocalized with MT1r and vice versa. Lack of TSH-producing cells accounted for absence of colocalization in seasonally anovulatory mares. The PT is considered a major target for melatonin, and colocalization with TSH supports a role for TSH as a modulator of seasonal reproduction in the mare. This is further evidenced by increased TSH immunosignal during the breeding season.

To understand the subcellular localization of RUNX2 and two lncRNAs, LINC02035 and LOC100130207, immunocytochemistry (for RUNX2 protein) and RNA _in situ_ hybridization assays (for both lncRNAs) were performed using human primary chondrocytes isolated from knee cartilage of OA patients. We confirmed that the RUNX2 protein was strongly detected in the nucleus of chondrocytes isolated from damaged cartilage (Figure 4A). The fractionated western blot results also showed that the RUNX2 protein was detected only in the nucleus of chondrocytes isolated from damaged cartilage (Figure 4B). To further understand the molecular mechanisms of the lncRNAs LINC02035 and LOC100130207, we performed an _in situ_ assay using primary chondrocytes derived from patients, because primary chondrocytes are a valuable model for studying OA pathogenesis. The results showed that both LINC02035 and LOC100130207 were highly expressed in chondrocytes isolated from the knee cartilage of patients with OA (Figure 4C). We then evaluated the mRNA levels and subcellular localization of both lncRNAs to elucidate their site of action using a commercially available kits in primary chondrocytes isolated from intact or damaged cartilage tissues. The results showed that both lncRNAs were more upregulated in primary chondrocytes isolated from damaged cartilage tissue than in intact cartilage tissue (Figure 4D). In primary chondrocytes, LINC02035 and LOC100130207 were merely detected in the cytoplasm of human primary chondrocytes and both lncRNAs were localized to nucleus (Figure 4E). Likewise, we also studied the subcellular localization of both lncRNAs in TC28a2 cells. The results showed that LINC02035 and LOC100130207 were evenly distributed in the nucleus and cytoplasm of normal chondrocytes (Figure 4F, left). However, both lncRNAs were preferentially localized to the nucleus and to a lesser extent to the cytoplasm after TC28a2 cells were treated with hypertrophic medium or TNF-α (Figure 4F, middle and right). To investigate whether RUNX2 is regulated at the post-translational level during hypertrophic changes in chondrocytes, human primary chondrocytes or TC28a2 cells were treated with the proteasome inhibitor MG132. The results showed that the protein level of RUNX2 was dose-dependently increased by MG132 treatment (Figure 4G-H), indicating that the upregulation of RUNX2 in osteoarthritic or hypertrophic chondrocytes occurs at the post-translational level. To examine whether both lncRNAs are involved in the stabilization of RUNX2 protein during hypertrophic differentiation and the inflammatory response in chondrocytes, IP was conducted to confirm the ubiquitination of RUNX2 protein. First, we investigated how the ubiquitination of RUNX2 protein is regulated during hypertrophic differentiation or the inflammatory response of chondrocytes, and as a result, it was confirmed that ubiquitination of RUNX2 was reduced by hypertrophic medium or TNF-α treatment (Figure 4I). However, ubiquitination of RUNX2 protein was clearly increased in TC28a2 cells transfected with siRNAs targeting LINC02035 or LOC100130207, even though the cells were treated with hypertrophic medium or TNF-α (Figure 4J-K). These results suggest that both lncRNAs upregulated during hypertrophic differentiation and the inflammatory response in chondrocytes contribute to the stabilization of the RUNX2 protein.