Disrupting the Indian hedgehog signaling pathway in vivo attenuates surgically induced osteoarthritis progression in Col2a1-CreERT2; Ihhfl/fl mice

Abstract

Introduction: Previous observations implicate Indian hedgehog (Ihh) signaling in osteoarthritis (OA) development because it regulates chondrocyte hypertrophy and matrix metallopeptidase 13 (MMP-13) expression. However, there is no direct genetic evidence for the role of Ihh in OA, because mice with cartilage or other tissue-specific deletion of the Ihh gene die shortly after birth. We evaluated the role of Ihh in vivo via a Cre-loxP-mediated approach to circumvent the early death caused by Ihh deficiency.

Methods: To evaluate the role of Ihh in OA development, Ihh was specifically deleted in murine cartilage using an Ihh conditional deletion construct (Col2a1-CreER(T2); Ihh(fl/fl)). The extent of cartilage degradation and OA progression after Ihh deletion was assessed by histological analysis, immunohistochemistry, real-time PCR and in vivo fluorescence molecular tomography (FMT) 2 months after OA was induced by partial medial meniscectomy. The effect of Ihh signaling on cartilage was compared between Ihh-deleted mice and their control littermates.

Results: Only mild OA changes were observed in Ihh-deleted mice, while control mice displayed significantly more cartilage damage. Typical OA markers such as type X collagen and MMP-13 were decreased in Ihh-deleted mice. In vivo FMT demonstrated decreased cathepsins and MMP activity in knee joints of animals with deletion of Ihh.

Conclusions: These findings support the protective role of Ihh deletion in surgically induced OA. Thus, our findings suggest the potential to develop new therapeutic strategies that can prevent and treat OA by inhibiting Ihh signaling in chondrocytes.

Figure 1

Col2a1-CreER^T2; Ihh^fl/fl mice exhibit normal phenotype before induction. (A) Insertion of loxP restriction enzyme site in the Indian hedgehog (Ihh) allele was confirmed by PCR. Lane 1: heterozygous Ihh^fl/fl (382 bp and 484 bp), lane 2: homozygous Ihh^fl/fl (382 bp), lane 3: wild type (WT) (484 bp), lane 4: 50-bp DNA marker and lane 5: Cre-recombinase (328 bp). (B) X-rays and Safranin O–stained images show normal growth plate and proteoglycan staining in WT mice (a) and mutant mice (b) at 2 months of age without deletion of Ihh.

Figure 2

Validation of Indian hedgehog deletion and partial medial meniscectomy. (A) Radiographs using soft-tissue settings show medial meniscus of the left knee joint without partial medial meniscectomy (PMM) surgery (indicated by arrowhead in left panel) and absence of medial meniscus of the right knee joint (indicated by arrow in right panel). (B) Results of real-time PCR are shown. Relative fold expression of Indian hedgehog (Ihh) is decreased by 46.5% in tamoxifen (TM)-injected mice. Data are means ± SD. TM, n = 6. no TM, n = 8. *P < 0.005.

Figure 3

Cathepsin and matrix metalloprotease activity were decreased in Indian hedgehog–deleted mice 2 months after partial medial meniscectomy. Representative images produced with ProSense (A) and MMPSense (B) fluorescent imaging agents (top panels) and summary results (bottom panels) are shown. Data are means ± SD. No tamoxifen–partial medial meniscectomy (TM-PMM), n = 12; TM-PMM, n = 8; and TM-sham, n = 3. *P = 0.016, #P = 0.042.

Figure 4

Deletion of Indian hedgehog alleviates osteoarthritis cartilage damage. (A) Coronal sections of mouse knee joints are shown after Safranin O staining. PMM, partial medial meniscectomy; TM, tamoxifen. (B) Osteoarthritis Research Society International Osteoarthritis Cartilage Histopathology Assessment System (OOCHAS) scores are shown. Data are means ± SD. No TM-PMM, n = 12; TM-PMM, n = 12; TM-Sham, n = 10. *P < 0.0001. In the control groups (No TM-Sham, n = 10; TM-Sham, n = 10; No TM-No Surgery, n = 8; TM-No Surgery, n = 8), there were no significant differences, #P = 0.585.

Figure 5

Indian hedgehog, matrix metalloprotease 13 and type X collagen expression is reduced in Ihh-deleted mice, but type II collagen and aggrecan are preserved. Immunohistochemical staining was used in osteoarthritis (OA)-induced tibial articular cartilage of 5-month-old mice for Indian hedgehog (Ihh) (A), matrix metalloprotease 13 (MMP-13) (B), type X collagen (Col X) (C), type II collagen (Col II) (D) and type II collagen breakdown products (Degraded Col II) (E). PMM, partial medial meniscectomy; TM, tamoxifen. Positive signals (brown staining) are indicated by arrows. Inside the black box was image taken under the 20x objective lens, and from which a small area was further enlarged under 20x objective lens (inside the red box)

Figure 6

Decreased Indian hedgehog signaling inhibits Ihh downstream targets in osteoarthritis cartilage. (A) Indian hedgehog (Ihh) deletion leads to downregulation of Ihh downstream targets in osteoarthritis (OA) cartilage 2 months after partial medial meniscectomy. (a) Laser-captured microdissection was used to analyze gene expression in articular cartilage. Articular cartilage (highlighted in red) before and after microdissection is shown. (b) Results of real-time PCR are shown. The expression of Ihh, Gli1, Gli2, matrix metalloprotease (MMP-13), type X collagen (Col X) and Runx2 was significantly decreased, and the expression of aggrecan and type II collagen (Col II) was increased, respectively, in the Ihh-deleted mice (tamoxifen (TM)) (n = 6) compared to the control animals (no TM) (n = 8). Data are means ± SD. *P < 0.00001, #P < 0.0005, +P < 0.001, ^P < 0.05. (B) Cyclopamine decreased expression of Gli1, MMP-13 and type X collagen in human OA cartilage. Relative mRNA levels in human cartilage organ cultures 48 hours after treatment with cyclopamine (n = 8) and dimethyl sulfoxide (DMSO) controls (n = 8) are shown. Data are means ± SD (n = 8). *#^P < 0.0001.