Deregulated proteolysis invariably underlies most human diseases including bone 
pathologies. Metalloproteinases constitute the largest of the five protease 
families, and the metzincin metalloproteinases are inhibited by the four tissue 
inhibitors of metalloproteinase called TIMPs. We hypothesized that Timp genes 
are essential for skeletal homeostasis. We bred individual Timp knockout mice to 
generate unique mouse models, the quadruple Timp null strain (QT) as well as 
mice harboring only a single Timp3 allele (QT3+/- ). QT mice are grossly smaller 
and exhibit a dramatic reduction of trabeculae in long bones by μCT imaging with 
a corresponding increase in metalloproteinase activity. At the cellular level, 
Timp deficiency compromised differentiation markers, matrix deposition and 
mineralization in neonatal osteoblasts from calvariae, as well as the 
fibroblastic colony-forming unit (CFU-F) capacity of bone marrow-derived stromal 
cells. In contrast, we observed that osteoclasts were overactive in the Timp 
null state, consistent with the noted excessive bone resorption of QT bones. 
Immunohistochemistry (IHC) and immunofluorescence (IF) analyses of bone sections 
revealed higher Cathepsin K and RANKL signals upon Timp loss. Seeking the 
molecular mechanism, we identified abnormal TNFα bioactivity to be a central 
event in Timp-deficient mice. Specifically, TNFα triggered induction of the Wnt 
signaling inhibitor Dkk1 in the osteoblasts at the mRNA and protein levels, with 
a simultaneous increase in RANKL. Neutralizing TNFα antibody was capable of 
rescuing the induction of Dkk1 as well as RANKL. Therefore, the generation of 
novel Timp-deficient systems allowed us to uncover the essential and collective 
function of TIMP proteins in mammalian long-bone homeostasis. Moreover, our 
study discovers a functional TIMP/metalloproteinase-TNFα-Dkk1/RANKL nexus for 
optimal control of the bone microenvironment, which dictates coexistence of the 
osteoblast and osteoclast lineages. © 2018 American Society for Bone and Mineral 
Research.