Epiphyseal bone formation occurs via thyroid hormone regulation of chondrocyte to osteoblast transdifferentiation

Abstract

Endochondral ossification in the diaphysis of long bones has been studied in-depth during fetal development but not postnatally in the epiphysis. Immunohistochemical studies revealed that Sox9 and Col2 expressing immature chondrocytes in the epiphysis transition into prehypertrophic and hypetrophic chondrocytes and finally into osteoblasts expressing Col1 and BSP during postnatal day 7-10, when serum levels of thyroid hormone (TH) rise. Lineage tracing using Rosa-td tomato ^{Col2-Cre-ERT2} mice treated with tamoxifen indicated that the same Col2 expressing chondrocytes expressed prehypertrophic, hypertrophic, and subsequently bone formation markers in a sequential manner in euthyroid but not hypothyroid mice, thus providing evidence that chondrocyte to osteoblast transdifferentiation is TH-dependent. Vascular invasion was apparent at the time of bone formation but not earlier. In vitro studies revealed that TH acting via TRα1 promoted expression of SHH while TRβ1 activation increased IHH but inhibited SHH expression. SHH promoted expression of markers of immature chondrocytes but inhibited chondrocyte hypertrophy while IHH promoted chondrocyte hypertrophy. Based on our data, we propose a model in which TH acting through TRα1 and TRβ1, respectively, fine tune levels of SHH and IHH and, thereby control the transit of proliferating immature chondrocytes into mature hypertrophic chondrocytes to become osteoblasts at the epiphysis.

Figure 1

The SOC begins ossification at post-natal Day 8 as shown by osteogenic and hypertrophic markers. (A-E’) P7, no SOC ossification can be seen, however, hypertrophic chondrocyte markers begin expression in the center of the SOC as denoted by MMP13. (F-J’) Ossification begins in the center of the SOC at a late P8 time point, hypertrophic cells surround forming osteoblasts (H), an earlier P8 time point shows lack of osteoid as can be seen in trichrome stain samples (G). (K-T’) P9 and P10 show continued expansion of SOC ossification. Hypertrophic CCs surround the periphery of the expanding SOC (M,R). ALP (N,S) and DMP1 (O,T) enlarge expression in the SOC. Yellow or black boxes enlarge related regions, “ ’ ” denotes enlarged region (e.g., A’). Large Scale bars are 100 μm and small scale bars are 200 μm.

Figure 2

The SOC matures, but does not become vascularized until after ossification as shown by vascularization and immature, prehypertrophic and hypertrophic chondrocyte marker expression. (A–E’) P7, the SOC has not yet formed and the center of the epiphysis expresses immature and prehypertrophic CC markers (SOX9, COL2, COL10, OSX), but is not vascularized. (F–J’) P8, immature CC markers disperse from the center of the epiphysis, hypertrophic markers expand. Vascularization through EMCN can be seen in the invagination at the periphery of the epiphysis by the articular CCs (J). (K–T’) P9 and P10 SOX9, COL2 and COL10 expression disperse further from the central epiphysis. EMCN expression can be seen as invading blood vessels in the SOC starting at P9 (N,N’, S,S’). Yellow boxes enlarge related regions, “ ’ ” denotes enlarged region (e.g., A’). Large Scale bars are 100 μm and small scale bars are 200 μm.

Figure 3

The bone begins to form prior to vascular invasion. (A-D) A time course analysis shows regions of bone formation denoted by BSPII (red) do not overlap with the invading vasculature visualized by EMCN (green) until the bone is already in the process of being formed. Time points indicate age and time of sacrifice. P8 6 am (A), P8 1 pm (B), P9 6 am (C), P9 6 pm (D). All sections are of proximal tibia except for (D) which displays the distal femur. (E,F) COL1 (red) co-stained with EMCN (green) shows a similar developmental pattern as the BSPII time course above. P8 1 pm (E) and P8 8 pm (F). (G,H) COL1 (red) co-stained with CD31 (green) indicates that the cells visualized by EMCN are indeed the invading vasculature. P8 1 pm (G) and P9 6 am (H). Yellow boxes enlarge related regions, “ ‘ ” denotes enlarged region (e.g., A’). “A” denotes significance. Large Scale bars are 100 μm and small scale bars are 200 μm.

Figure 4

SOC osteogenic tissue arises from Col2 + chondrocytes (A–I’) Lineage trace analysis of Col2-Cre ^ERT2 mice reveals that most cells in the epiphysis express TOM. As cells become hypertrophic (MMP13) (A,E) they continue to express TOM. Eventually, hypertrophic cells surround unlabeled TOM + cells (I). TOM + cells do, however, co-label with osteogenic markers such as ALP (F,J), DMP1 (G,K) and COL1 (H,L) at P9 and P10 during SOC formation. (M,N) Later stage, 12 week (12 W) Col2-Cre ^ERT2 mice have SOCs which contain a large amount of TOM + cells as well as those colabeled with OSX (M; arrow) and ALP (N; arrow). (O) Methimazole treated Col2-Cre ^ERT2 mice did not form an SOC and only expressed low levels of DMP1 by P14. The SOC is usually well into its formation at this time point. Yellow boxes enlarge related regions, “ ‘ ” denotes enlarged region (e.g., A’). Large Scale bars are 100 μm and small scale bars are 200 μm.

Figure 5

Thyroid hormone is required for CC hypertrophy and osteogenesis in the epiphysis. (A–T’) Hyt/hyt mice do not form an SOC between P7-P10 as confirmed by safranin-O (A,F,K,P), trichrome (B,G,L,Q), lack of CC hypertrophy via MMP13 (C, H, M, R), and lack of osteogenic expression via ALP (D,I,N,S) and DMP1 (E,J,O,T). Yellow or black boxes enlarge related regions, “ ’ ” denotes enlarged region (e.g., A’). Large Scale bars are 100 μm and small scale bars are 200 μm.

Figure 6

CCs do not mature and no vascularization occurs in the epiphysis in the absence of thyroid hormone. (A–T’) Between P7-P10 hyt/hyt mice do not form mature epiphyseal CCs as seen by high expression of SOX9 (A,F,K,P) and COL2 (B,G,L,Q), remained prehypertrophic via COL10 (C, H, M,R), and lack of osteogenic expression via OSX (D,I,N,S). Further, no vascular invasion was detected via EMCN (E,J,O,T). Yellow boxes enlarge related regions, “ ’ ” denotes enlarged region (e.g., A’). Large Scale bars are 100 μm and small scale bars are 200 μm.

Figure 7

Thyroid hormone acts through THRβ1 and HH expression to influence epiphyseal chondrocyte differentiation. (A–D) IHC data confirms a higher IHH expression in the SOC vs SHH (green) in P7 hyt/ + mice (A,C), whereas there is an extreme reduction of IHH expression coupled with an increase in SHH expression in hyt/hyt mice (B,D). (E–H) IHC data indicate unchanged THRα1 levels in both hyt/ + and hyt/hyt animals (E,F). Conversely, THRβ1 expression is markedly reduced in hyt/hyt compared to hyt/ + animals (G,H). (I,J) Ihh mRNA expression of P7 epiphyses decreased 7 fold while Shh expression is increased 2.4 fold in hyt/hyt vs hyt/ + animals (I). P7 epiphyses of hyt/hyt animals showed a 2.2 fold decrease in Thrβ1 but no effect in Thrα1 expression compared to hyt/  ⁺animals (J). (K) Thrβ1 knockdown in cultured CCs increased Shh expression 3.4 fold and reduced Ihh expression 2 fold. (L) Adenoviral OE of Shh in epiphyseal CCs increased Sox9, Col2, and Acan mRNA expression (1.5, 3.5, and 3 fold, respectively). Mmp13 and Rankl expression decrease 1.7 and 2.8 fold, respectively. Ptch1 and Gli1 expression is increased 9 and 13.5 fold, respectively. Gli2 and Gli3 expression remain unchanged. All mRNA is tested against GFP adenovirally treated cells. (M, N) shRNA knockdown of Ihh in ATDC5 CCs causes an increase in Gli1, however, Ptch1 and Gli2 are knocked down (2 fold each) (M) It also reduces Mmp13 and Bsp expression (5 and 3.3 fold, respectively) but increases Col2 expression (2.5 fold) (N). Red Circles denote the SOC initiation region. All RNA data are represented as a mean ± SEM, “A” denotes significance. Large Scale bars are 100 μm and small scale bars are 200 μm.

Figure 8

Model for early postnatal development of the SOC. During embryonic and early postnatal development when TH levels are low, epiphyseal chondrocytes express high levels of SHH which acts though Gli1 to maintain these cells in proliferative immature state by activating Sox5/6/9 transcriptional activity. At P6/P7, rise in TH increases THRβ1expression and thereby IHH expression. IHH acts through GLI2 to decrease SOX9 and COL2 expression, while MMP13 and ADAMTS5 expression increases to deplete the COL2 matrix. Pre-hypertrophic CCs begin to express COL10 and OSX in the P8/P9 period. These in turn activate DMP1 and ALP in the SOC, meanwhile blood vessels invade from the periphery of the articular CCs.