Primary cilia modulate Ihh signal transduction in response to hydrostatic loading of growth plate chondrocytes

Abstract

Indian hedgehog (Ihh) is a key component of the regulatory apparatus governing chondrocyte proliferation and differentiation in the growth plate. Recent studies have demonstrated that the primary cilium is the site of Ihh signaling within the cell, and that primary cilia are essential for bone and cartilage formation. Primary cilia are also postulated to act as mechanosensory organelles that transduce mechanical forces acting on the cell into biological signals. In this study, we used a hydrostatic compression system to examine Ihh signal transduction under the influence of mechanical load. Our results demonstrate that hydrostatic compression increased both Ihh gene expression and Ihh-responsive Gli-luciferase activity. These increases were aborted by disrupting the primary cilia structure with chloral hydrate. These results suggest that growth plate chondrocytes respond to hydrostatic loading by increasing Ihh signaling, and that the primary cilium is required for this mechano-biological signal transduction to occur.

Figure 1. Custom-designed hydrostatic loading system

The module consists of a PMMA/aluminum (A, red arrows) frame surrounding the bioreactor chamber (A, green arrow) with the intervening space filled with water (B, dark blue). When pressuring the bioreactor, the media ports are closed using valves (A, blue arrows), and pressure is applied to the sample across the flexible FEP membrane (B, arrows). A perforated insert holds up to nine aggregates. Automatic control was achieved by using custom-written C software running on an IBM-PC. This module allows the application of arbitrary hydrostatic pressure waveforms on the construct in the bioreactor.

Figure 2. Proliferation Assay

Aggregates either were left unloaded (Control) or were hydrostatically loaded for 24 or 48 hours as described (Compressed). Immediately after compression, the cells were incubated at 37 °C in 200 µl of medium with 40 µl of the MTS/PMS solution (from the Titer AQ kit) for 1 hour. Absorbance was read at 490 nm in a 96-well plate reader. Data are shown normalized to the control group (* indicates p = 0.0005).

Figure 3. Effects of hydrostatic compression loading on Ihh gene expression in growth plate chondrocytes

Unloaded (Control), and compressed samples were collected after either A: 1 (*p = 0.014), or 2 days (*p = 0.0002) or B: After 0, 0.5, 1, 2, and 4 hours of intermittent hydrostatic compression (p =0.017 by ANOVA, * p = 0.017, 0.03, 0.02 respectively).

Figure 4. Gli-dependent luciferase activity

Gli-luciferase reporter construct was transiently transfected into primary growth plate chondroctyes, which were then cultured as cell pellets. A: Luciferase activity was measured after 48 hours of treatment without (Control) or with compression (Compression, * p = 0.04). The compression induced increase in activity was abolished by the addition of 10 nM cyclopamine (Compressed + cyclopamine). Four mM chloral hydrate essentially eliminated Gli-luciferase activity either without (Chloral hydrate, * p = 0.00002); or with hydrostatic compression (Compressed + chloral hydrate, * p = 0.00002); B: Cyclopamine abolishes the Shh-induced increase in Gli expression. Gli-Luciferase activity was measured after 48 hours of treatment with or without 5nM sonic hedgehog (Shh, * p = 0.006 compared to control), Shh + Cyclopamine, or Cyclopamine alone (* p < 0.05, compare to Shh).

Figure 5. Cilia structure in growth plate chondrocytes with or without chloral hydrate treatment

Confocal image of primary cilia (arrows) immunostained with an anti-acetylated α-tubulin antibody. A: Control; B: After 2 days of chloral hydrate (4 mM) treatment. Scale bar = 10µm.

Figure 6. Ihh and Smo expressions were affected by chloral hydrate

A: Chloral hydrate treatment significantly reduced mRNA level (measured by real time PCR) of Ihh (* p = 0.012) and Smo (* p = 4 × 10⁻⁶), Shh treatment significantly (* p = 10⁻²) increased Smo, but not Ihh expression, and the increase was abolished by chloral hydrate treatment (* p = 10⁻⁶). B: Protein levels of Ihh and Smo were also significantly reduced (* p = 0.027 and 0.001, respectively) as measured by Western blotting.