Sonic Hedgehog influences the balance of osteogenesis and adipogenesis in mouse adipose-derived stromal cells

Abstract

Adipose-derived stromal cells (ASCs) present a great potential for tissue engineering, as they are capable of differentiating into osteogenic and adipogenic cell types, among others. In this study, we examined the role of Hedgehog signaling in the balance of osteogenic and adipogenic differentiation in mouse ASCs. Results showed that Hedgehog signaling increased during early osteogenic differentiation (Shh, Ptc1, and Gli1), but decreased during adipogenic differentiation. N-terminal Sonic Hedgehog (Shh-N) significantly increased in vitro osteogenic differentiation in mouse ASCs, by all markers examined (*p < 0.01). Concomitantly, Shh-N abrogated adipogenic differentiation, by all markers examined (*p < 0.01). Conversely, blockade of endogenous Hedgehog signaling, with the Hedgehog antagonist cyclopamine, enhanced adipogenesis at the expense of osteogenesis. We next translated these results to a mouse model of appendicular skeletal regeneration. Using quantitative real-time polymerase chain reaction and in situ hybridization, we found that skeletal injury (a monocortical 1 mm defect in the tibia) results in a localized increase in Hedgehog signaling. Moreover, grafting of ASCs treated with Shh-N resulted in significantly increased bone regeneration within the defect site. In conclusion, Hedgehog signaling enhances the osteogenic differentiation of mouse ASCs, at the expense of adipogenesis. These data suggest that Hedgehog signaling directs the lineage differentiation of mesodermal stem cells and represents a promising strategy for skeletal tissue regeneration.

FIG. 1.

Hedgehog signaling during osteogenic and adipogenic differentiation. (A) Progression of mouse adipose-derived stromal cell (ASC) osteogenic differentiation through 2 weeks, shown by alkaline phosphatase (ALP) and alizarin red (AR) staining. (B) Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), Gli1, and patched (Ptc1) expression at 0, 1, and 2 weeks of osteogenic differentiation, normalized to GAPDH. (C) Mouse ASC adipogenic differentiation, shown by oil red O staining. (D) Shh, Ihh, Gli1, and Ptc1 expression at 0 and 1 week of adipogenic differentiation, normalized to GAPDH. Photographs are of 20× magnification; means and significance levels are calculated relative to 0 week expression, ^a,b,c,dp < 0.01. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Hedgehog pathway activation and adipogenic differentiation in ASCs. (A) Hedgehog signaling elements following culture with N-terminal Sonic Hedgehog (Shh-N) for 48 h by quantitative real-time polymerase chain reaction (qRT-PCR), including Ptc1, Gli1, Gli2, and Gli3. (B) Hedgehog ligand expression after Shh-N exposure for 48 h, including Shh, Ihh, and Dhh. (C–F) Adipogenic differentiation with Shh-N or cyclopamine. (C) Oil red O staining with Shh-N at day 7 adipogenic differentiation (250–750 ng/mL). (D) Oil red O staining with cyclopamine at day 7 differentiation (5–20 μM). (E) PPAR-γ expression with Shh-N at 7 days, by qRT-PCR. (F) PPAR-γ expression with cyclopamine at 7 days of differentiation. Means and significance levels are calculated relative to control expression levels, ^a,b,c,dp < 0.01. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Osteogenic differentiation with Shh-N. Osteogenic differentiation was examined through a 14 day time course. (A) Runx2 and collagen type I (Col1) expression by qRT-PCR after 4 days. (B) ALP staining after 7 days, appearing blue. ALP quantification after 7 days was normalized to total protein content. (C) AR staining and quantification after 14 days. (D) Osteopontin (Opn) and Osteocalcin (Oc) expression by qRT-PCR after 14 days. Photographs are of 20× magnification; values are normalized to control expression levels, ^ap < 0.01. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Osteogenic differentiation of ASCs with cyclopamine. (A) Runx2 and Col1 expression with cyclopamine and/or Shh-N (500 ng/mL) at 4 days of differentiation, by qRT-PCR. (B) ALP staining and quantification with cyclopamine and/or Shh-N after 7 days. (C) AR staining and quantification with cyclopamine and/or Shh-N after 14 days. Photographs are of 20× magnification; values are normalized to control expression levels, ^a,b,cp < 0.01. Color images available online at www.liebertonline.com/ten.

FIG. 5.

Cooperation of Hedgehog and bone morphogenetic protein (BMP) signaling. (A) Bmp2 and Bmp4 expression after 48 h of Shh-N treatment, by qRT-PCR. (B) Hedgehog pathway activity after 48 h of recombinant (r)BMP-2 treatment, by qRT-PCR. (C) ALP staining (left) and quantification (right) after 7 days with Shh-N and/or rBMP-2. Bars represent mean ALP enzymatic activity, normalized to total protein content. (D) AR staining (left) and quantification (right) after 14 days with Shh-N and/or rBMP-2. Bars represent mean AR staining, as measured by absorbance after cetylpyridium chloride leaching. Photographs are of 20× magnification; values are normalized to control expression levels, ^a,bp < 0.01. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Grafting of ASCs in tibial defects. (A, B) A 1-mm monocortical defect was surgically created in the proximal mouse tibia; spatiotemporal gene expression after injury was then examined by qRT-PCR and in situ hybridization. (A) Hedgehog signaling expression from 1 to 6 days postinjury (Gli1, Smo, and Ptc1). (B) Ptc1 in situ in which positive staining cells appear purple; dotted lines demarcate the cortical bone. (C) Schematic of the grafting of green fluorescent protein (GFP)–positive ASCs into a monocortical tibial defect. (D, E) Histology of defects with Shh-N- or control-treated ASCs. From top to bottom, green fluorescent protein immunohistochemistry appearing gray, ALP staining appearing purple, pentachrome staining in which bone appears yellow, and phospho-Smad 1/5/8 immunohistochemstry appearing brown. Black arrows D indicate examples of positively stained cells for phospho-Smad 1/5/8. Color images available online at www.liebertonline.com/ten.