The alchemy of tendon repair: A primer for the (S)mad scientist

During vertebrate development, mesenchymal progenitors capable of forming bone, cartilage, muscle, fat, or tendon arise from either neural crest or somitic mesoderm. Transcriptional programs that specify mesenchymal cell fates are initiated and modified by paracrine cues provided by TGF-β superfamily members and mediated in part via the regulated assembly of Smad-containing multiprotein transcription factor complexes. In this issue of the JCI, Hoffmann and colleagues have identified that Smad8 activation drives tendon formation from C3H10T1/2 cells, a murine cell line that recapitulates many features of normal multipotent mesenchymal cells (see the related article beginning on page 940). Cells programmed to the tenocyte cell fate in vitro formed tenogenic grafts in vivo. These results add to the accumulating evidence that proliferating, multipotent mesenchymal progenitor cells can be programmed to yield multiple cell types — e.g., osteoblasts, myocytes, chondrocytes, and tenocytes — that may be useful in cell-based therapeutic approaches to musculoskeletal diseases

Runx2 Trans-Activation Mediated by the Msx2-Interacting Nuclear Target Requires Homeodomain Interacting Protein Kinase-3

Runt-related transcription factor 2 (Runx2) and muscle segment homeobox homolog 2-interacting nuclear target (MINT) (Spen homolog) are transcriptional regulators critical for mammalian development. MINT enhances Runx2 activation of osteocalcin (OC) fibroblast growth factor (FGF) response element in an FGF2-dependent fashion in C3H10T1/2 cells. Although the MINT N-terminal RNA recognition motif domain contributes, the muscle segment homeobox homolog 2-interacting domain is sufficient for Runx2 activation. Intriguingly, Runx1 cannot replace Runx2 in this assay. To better understand this Runx2 signaling cascade, we performed structure-function analysis of the Runx2-MINT trans-activation relationship. Systematic truncation and domain swapping in Runx1:Runx2 chimeras identified that the unique Runx2 activation domain 3 (AD3), encompassed by residues 316–421, conveys MINT+FGF2 trans-activation in transfection assays. Ala mutagenesis of Runx2 Ser/Thr residues identified that S301 and T326 in AD3 are necessary for full MINT+FGF2 trans-activation. Conversely, phosphomimetic Asp substitution of these AD3 Ser/Thr residues enhanced activation by MINT. Adjacent Pro residues implicated regulation by a proline-directed protein kinase (PDPK). Systematic screening with PDPK inhibitors identified that the casein kinase-2/homeodomain-interacting protein kinase (HIPK)/dual specificity tyrosine phosphorylation regulated kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), but not ERK, c-Jun N-terminal kinase, p38MAPK, or other casein kinase-2 inhibitors, abrogated Runx2-, MINT-, and FGF2-activation. Systematic small interfering RNA-mediated silencing of DMAT-inhibited PDPKs revealed that HIPK3 depletion reduced MINT+FGF2-dependent activation of Runx2. HIPK3 and Runx2 coprecipitate after in vitro transcription-translation, and recombinant HIPK3 recognizes Runx2 AD3 as kinase substrate. Furthermore, DMAT treatment and HIPK3 RNAi inhibited MINT+FGF2 activation of Runx2 AD3, and nuclear HIPK3 colocalized with MINT. HIPK3 antisense oligodeoxynucleotide selectively reduced Runx2 protein accumulation and OC gene expression in C3H10T1/2 cells. Thus, HIPK3 participates in MINT+FGF2 regulation of Runx2 AD3 activity and controls Runx2-dependent OC expression