Bone dysplasia in Hutchinson-Gilford progeria syndrome is associated with dysregulated differentiation and function of bone cell populations

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder affecting tissues of mesenchymal origin. Most individuals with HGPS harbor a de novo c.1824C > T (p.G608G) mutation in the gene encoding lamin A (LMNA), which activates a cryptic splice donor site resulting in production of the toxic "progerin" protein. Clinical manifestations include growth deficiency, lipodystrophy, sclerotic dermis, cardiovascular defects, and bone dysplasia. Here we utilized the Lmna^G609G knock-in (KI) mouse model of HGPS to further define mechanisms of bone loss associated with normal and premature aging disorders. Newborn skeletal staining of KI mice revealed altered rib cage shape and spinal curvature, and delayed calvarial mineralization with increased craniofacial and mandibular cartilage content. MicroCT analysis and mechanical testing of adult femurs indicated increased fragility associated with reduced bone mass, recapitulating the progressive bone deterioration that occurs in HGPS patients. We investigated mechanisms of bone loss in KI mice at the cellular level in bone cell populations. Formation of wild-type and KI osteoclasts from marrow-derived precursors was inhibited by KI osteoblast-conditioned media in vitro, suggesting a secreted factor(s) responsible for decreased osteoclasts on KI trabecular surfaces in vivo. Cultured KI osteoblasts exhibited abnormal differentiation characterized by reduced deposition and mineralization of extracellular matrix with increased lipid accumulation compared to wild-type, providing a mechanism for altered bone formation. Furthermore, quantitative analyses of KI transcripts confirmed upregulation of adipogenic genes both in vitro and in vivo. Thus, osteoblast phenotypic plasticity, inflammation and altered cellular cross-talk contribute to abnormal bone formation in HGPS mice.

Keywords: Hutchinson-Gilford progeria syndrome; bone dysplasia; lamin A/C; osteoblasts; osteoclasts.

FIGURE 1

Abnormal bone structural parameters in Lmna ^G609G mice. (a) Eight‐week old homozygous (G/G) mice have significantly shorter femora and tibiae compared to wild‐type (+/+) and heterozygous (G/+) littermates. Shortening of femora in homozygous (G/G) mice is relatively greater than in tibiae resulting in rhizomelia. N = 10 for each gender and genotype; *p < 0.05; **p < 0.01; ***p < 0.001 (b) H&E staining reveals disorganization of distal growth plates and femoral head, especially in homozygous (Lmna ^G609G/G609G) mice at 2 months of age. TUNEL staining of whole femora detects increased DNA damage in distal growth plate and femoral head chondrocytes in heterozygous (Lmna ^G609G/+) and homozygous (Lmna ^G609G/G609G) mice.

FIGURE 2

Expression of progerin alters femoral bone structural and material properties. (a) Reconstructed images from microCT analysis illustrate reduced trabecular and cortical bone volume in heterozygous (Lmna ^G609G/+) and homozygous (Lmna ^G609G/G609G) compared to wild‐type femora at 2 and 8 months of age. (b) Abnormal collagen cross‐link profiles are observed in 2 month‐old heterozygous (G/+) and homozygous (G/G) mice. Immature, divalent lysine‐derived (DHLNL: reduced dehydro‐dihydroxylysinonorleucine/it's ketoamine) and hydroxylysine‐derived (HLNL: reduced dehydro‐hydroxylysinonorleucine/it's ketoamine) crosslinks are increased relative to wild‐type mice. Total collagen aldehydes, catalyzed by LOX activity, are increased in progeroid mice. *p < 0.05, **p < 0.01, ***p < 0.001 versus Lmna ^+/+ littermates (c) Total collagen content and extent of lysine hydroxylation (Hyl) are normal in Lmna ^G609G/+ (G/+) and Lmna ^G609G/G609G (G/G) versus wild‐type (+/+) bone. N = 3/genotype.

FIGURE 3

Expression of progerin alters signaling pathways required for osteogenesis. (a) Quantitation of A‐ and B‐type lamin transcripts in femoral tissue of 2‐month mice. Expression of B‐type lamins, Lmnb1 (B1) and Lmnb2 (B2) remains stable regardless of genotype. (b) Western analysis of A‐type lamins extracted from bone tissue and osteoblast cultures derived from wild‐type (Lmna ^+/+, +/+), heterozygous (Lmna ^G609G/+, G/+), and homozygous (Lmna ^G609G/G609G, G/G) mice. (c) Gene set enrichment analysis of KEGG Pathways reveals positive enrichment for metabolic and cytokine activity and negative enrichment for cell–cell and cell‐matrix interactions. (d) Type I procollagen N‐propeptide (PINP), a marker of bone‐forming activity of osteoblasts, is decreased in serum of heterozygous (G/+) and homozygous (G/G) mice at 2 months of age and in heterozygous (G/+) mice at 8 months of age compared to wild‐type mice. N = 12 per genotype (six males, six females). (e) Marrow lipid content of heterozygous (G/+) and homozygous (G/G) mice at 2 months of age is increased versus wild‐type. N = 8 per genotype (four males, four females). *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

Expression of A‐type lamins in cultured murine osteoblasts. (a) Quantitative RT‐PCR of lamin A (A), progerin (P), and lamin C (C) transcripts from cultured osteoblasts at days 1, 5, 10, 15, 20, and 30 of osteogenic differentiation. (b) Immunoblots of cellular protein show increased accumulation of A‐type lamins at late stages of differentiation. (c) Left, Immunostaining demonstrates localization of A‐type lamins within the nucleus of cultured osteoblasts. Cells were stained to visualize nuclei (DAPI, blue), Actin (red), and A‐type lamins (green, antibody 4c11). Right, progerin‐specific antibody (Y288) reacts with the nucleoplasm, nuclear lamina, and cytoplasmic elements in cultured Lmna ^G609G/G609G (Lmna^G/G) osteoblasts. Cells were stained to visualize nuclei (DAPI, blue), Actin (green), and progerin (red). Scale bars, 50 μm at 20× magnification; 15 μm at 60× magnification.

FIGURE 5

Increased phenotypic plasticity in Lmna ^G609G/G609G osteoblasts. (a) Left, newborn calvarial pre‐osteoblasts isolated from homozygous (Lmna ^G609G/G609G) mice exhibit reduced capacity to deposit and mineralize matrix compared to wild‐type (Lmna ^+/+) and heterozygous (Lmna ^G609G/+) cultures. Cells were maintained in osteogenic media for 3 weeks followed by staining with alizarin red. Right, homozygous cultures accumulate lipids, detected by oil red‐O staining, when cultured in adipogenic media for 2 weeks. (b) Quantitative RT‐PCR of transcripts from differentiating Lmna ^G609G/G609G osteoblasts shows dysregulated expression of osteogenic markers required for differentiation, cellular crosstalk, and matrix mineralization. Results are the averages of three independent experiments. (c) Lmna ^G609G/G609G osteoblasts express higher levels of Pparg2 transcripts, left, and PPARG protein at earlier stages of differentiation compared to wild‐type (Lmna ^+/+) cells, right. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 6

Osteoclastogenesis is affected by Lmna ^G609G/G609G osteoblast secreted factors. (a) Serum levels of TRAcP are increased in 2 month‐old homozygotes (G/G) compared to wild‐type (+/+) and heterozygous (G/+) mice. N = 12/genotype; 6 males, 6 females. *p < 0.05; **p < 0.01; ***p < 0.001 (b) Serum Type I collagen C‐telopeptide levels (CTX) are equivalent in 2 and 8 month‐old mice. N = 12/genotype; 6 males, 6 females. (c) Quantitation of Lmna‐derived transcripts in cultured marrow‐derived osteoclasts following treatment with MCSF and RANKL to induce fusion, including Lmna (A), Progerin (P), and Lmnc (C) transcripts. (d) Immunoblot of cultured mononuclear precursors (treated with MCSF) and mature, multinucleated osteoclast (treated with MCSF+RANKL) lysates demonstrates the presence of LMNA, Progerin, and LMNC. A‐type lamins are observed at greater levels in multinucleated osteoclasts compared to mononuclear precursors. (e) Wide‐field imaging of marrow‐derived mononuclear precursors (MCSF) and multinucleated osteoclasts (MCSF+RANKL) at 20× magnification. Cells were stained to visualize nuclei (DAPI, blue), Actin (green), and progerin (red). (f) Static histomorphometric analysis of osteoclasts on femoral trabecular surfaces of 8 week‐old wild‐type (Lmna^+/+) and homozygous (Lmna^G/G) mice demonstrates reduced osteoclast surface (Oc. S), osteoclast surface per bone surface (Oc. S/BS) and osteoclast number per bone surface (Oc. N/BS). N = 12 per genotype (six males, six females). (g) Immunofluorescent staining of cultured mature osteoclasts derived from wild‐type (Lmna ^+/+) and homozygous (Lmna ^G/G) 8‐week marrow progenitors in the presence of femoral osteoblast‐conditioned media. The number of nuclei present in osteoclasts cultured under each condition was obtained from two independent cultures. N, number of cells analyzed.