Faculty Bibliography

Eph receptors belong to a subfamily of receptor tyrosine kinases that are activated by membrane-spanning ligands called ephrins. Previously, we demonstrated that the ephrinB1-EphB2 interaction regulates odontogenic/osteogenic differentiation from dental pulp cells (DPCs) in vitro. The goal of this study was to identify the molecular mechanisms regulated by the EphB2/ephrinB1 system that govern tertiary dentin formation in vitro and in vivo. During tooth development, ephrinB1, and EphB2 were expressed in preodontoblast and odontoblasts at postnatal day 4. EphrinB1 was continuously expressed in odontoblasts and odontoblastic processes until the completion of tooth eruption. In addition, ephrinB1 was expressed in odontoblastic processes 2 wk following tooth injury without pulp exposure, whereas EphB2 was expressed in the center of pulp niches but not odontoblasts. In a model of tooth injury with pulp exposure, ephrinB1 was strongly expressed in odontoblasts 4 wk postinjury. In vitro studies with human and mouse DPCs treated with calcium hydroxide (CH) or mineral trioxide aggregate (MTA) showed an increased expression of insulin-like growth factor 1 (IGF-1). Experiments using several inhibitors of IGF-1 receptor signaling revealed that inhibiting the Ras/Raf-1/MAPK pathway inhibited EphB2 expression, and inhibiting the PI3K/Akt/mTOR pathway specifically inhibited ephrinB1 gene expression. Tooth injury in mice with odontoblast-specific IGF-1 receptor ablation exhibited a reduced tertiary dentin volume, mineral density, and ephrinB1 expression 4 wk following injury. We conclude that the IGF-1/ephrinB1 axis plays significant roles in the early stages of tooth injury. Further research is needed to fully understand the potential of targeting ephrinB1 as a regenerative pulp therapy.

Growth hormone (GH) and insulin like growth factor-1 (IGF-1) are anabolic hormones that facilitate somatic and skeletal growth, and regulate metabolism via endocrine and autocrine/paracrine mechanisms. We hypothesized that excess tissue production of GH will protect skeletal growth and integrity in states of reductions in serum IGF-1 levels. To test our hypothesis we used the bovine GH (bGH) transgenic mice as a model of GH hypersecretion and ablated the liver-derived acid labile subunit (ALS), which stabilizes IGF-1 complexes with IGF-binding protein-3 (IGFBP-3) and -5 in circulation. We used a genetic approach to create bGH/ALSKO mice, and siRNA gene silencing approach to reduce als or igf-1 gene expression. We found that in both models, decreased IGF-1 levels in serum associated with decreased body and skeletal size of the bGH mice. Excess GH produced more robust bones, but compromised mechanical properties in male mice. Excess GH production in tissues did not protect from trabecular bone loss in response to reductions in serum IGF-1 (in bGH/ALSKO or bGH mice treated with siRNAs). Reduced serum IGF-1 levels in the bGH mice did not alleviate the hyperinsulinemia, and did not resolve liver or kidney pathologies that resulted from GH hypersecretion. We conclude that reduced serum IGF-1 levels decrease somatic and skeletal growth even in states of excess GH.

Mutation in the insulin-like growth factor-1 receptor (IGF1R) gene is a rare cause for intrauterine and postnatal growth disorders. Patients identified with IGF1R mutations present with either normal or impaired glucose tolerance. None of the cases described so far showed hypoglycemia. We aimed to identify the genetic basis for small for gestational age, short stature and hypoglycemia over three generations in one family. The proband, a 9-year-old male, presented in infancy with recurrent hypoglycemic episodes, symmetric intrauterine growth retardation and postnatal growth retardation. Blood DNA samples from the patient, his parents, a maternal sister and maternal grandmother underwent Sanger sequencing of the IGF1R gene. Primary skin fibroblast cultures of the patient, his mother and age- and sex-matched control donors were used for gene expression and receptor functional analyses. We found a novel heterozygous mutation (c.94 + 1g > a, D1105E) affecting the splicing site of the IGF1R mRNA in the patient, his mother and his grandmother. Primary fibroblast cultures derived from the patient and his mother showed reduced proliferation and impaired activation of the IGF1R, evident by reduced IGF1R and AKT phosphorylation upon ligand binding. In conclusion, the newly identified heterozygous missense mutation in exon 1 of IGF1R (D1105E) results in impaired IGF1R function and is associated with small for gestational age, microcephaly and abnormal glucose metabolism. Further studies are required to understand the mechanisms by which this mutation leads to hypoglycemia.

Eph receptors belong to a subfamily of receptor tyrosine kinases activated by membrane bound ligands called ephrins. Recent studies have shown that osteoblasts express the EphB4 receptor and it's ligand ephrinB2 and B1, while osteoclasts express a few members of the ephrinBs, suggesting that these proteins are involved in bone modeling during growth. Past studies have shown that parathyroid hormone (PTH) stimulates bone modeling via enhanced expression of the insulin-like growth factor-1 (IGF-I) and the ephrinB2 and EphB4 in osteoblasts. To define the interactions between PTH/IGF-1/Eph signaling pathways in bone, male mice at 4 weeks of age were injected 80 mcg/kg/day for 5 days. To our surprise we found 3 fold increase in the expression of Ephrin B1 in the femoral cortical shells from male mice, while the expression of ephrinB2 or EphB4 did not differ significantly between PTH treated and untreated groups. Thus, we used the osteoblast- and osteocyte-specific ephrinB1 knockout (KO) mice (using the osteocalcin-cre and the dentin matrix protein (DMP)-1-cre, respectively) to investigate their bone response to intermittent PTH treatment. We used micro Computer Tomography (CT) to characterize the basal morphology of femurs dissected from male mice. We found decreases in tissue mineral density in cortical bone of the mid-shaft femur in both Osteocalcin-ephrinB1 (1.45 +/- 0.05 g/cm³ , p=0.01) and DMP-1-ephrinB1 KO mice (1.465 +/- 0.05 mg/cm³ , p=0.02) as compared to controls (1.64 +/- 0.02 mg/cm³ ) . Similarly, we found decreased bone mineral density in the trabecular bone assessed at the distal femur (Osteocalcin-eprinB1 0.147+/-0.005 p=0.004, DMP-1-ephrinB1 0.175 +/- 0.02 p=0.06, and controls 0.248 +/- 0.02 mg/cm³ ) . However, cortical and trabecular bone morphology of males did not differ between the groups. Female DMP-ephrinB1 KO mice showed decreased total cross-sectional area (1.14 +/- 0.09 vs 1.27+/-0.06 mm² , p = 0.02), polar moment of inertia (0.12 +/- 0.03 vs 0.14+/- 0.01 mm⁴ , p= 0.03), and marrow area (0.76 +/- 0.03 vs 0.86 +/- 0.06 mm² , p = 0.03) as compared to control mice with no significant difference in tissue mineral density. Female DMPephrinB1 KO mice did not show any trabecular bone phenotype. Bone anabolic response to intermittent PTH treatment in Osteocalcin-eprinB1 and DMP-1-ephrinB1 KO mice is under investigation

Type 1 diabetes mellitus (T1DM) is one of the most common chronic diseases diagnosed in childhood. Childhood and adolescent years are also the most important period for growth in height and acquisition of skeletal bone mineral density (BMD). The growth hormone (GH)/insulin like growth factor -1 (IGF-1) axis which regulates growth, is affected by T1DM, with studies showing increased GH and decreased IGF-1 levels in children with T1DM. There is conflicting data as to whether adolescents with TIDM are able to achieve their genetically-determined adult height. Furthermore, data support that adolescents with T1DM have decreased peak BMD, although the pathophysiology of which has not been completely defined. Various mechanisms have been proposed for the decrease in BMD including low osteocalcin levels, reflecting decreased bone formation; increased sclerostin, an inhibitor of bone anabolic pathways; and increased leptin, an adipocytokine which affects bone metabolism via central and peripheral mechanisms. Other factors implicated in the increased bone resorption in T1DM include upregulation of the osteoprotegerin/ receptor-activator of the nuclear factor-kappaB ligand pathway, elevated parathyroid hormone levels, and activation of other cytokines involved in chronic systemic inflammation. In this review, we summarize the clinical studies that address the alterations in the GH/IGF-I axis, linear growth velocity, and BMD in children and adolescents with T1DM; and we review the possible molecular mechanisms that may contribute to an attenuation of linear growth and to the reduction in the acquisition of peak bone mass in the child and adolescent with T1DM.

Mitochondrial dysfunction has been recognized as a prominent feature of the diminishing cell function in aging bone. Studies of the long-lived growth hormone receptor knockout (GHRKO) mice, which show compromised skeletal growth, have suggested increased mitochondrial biogenesis and function in tissues, such as kidney, heart, and skin fibroblasts. The roles of GHR in maintaining mitochondrial function in osteocytes, the predominant population of bone cells, were not studied. Our goal was to understand the mechanisms by which GHRKO affects mitochondrial volume and function in the adult and aged bones. Using primary osteocyte cultures from 8 weeks old control and GHRKO mice we found that unlike in kidney and heart, in bone tissue of GHRKO mice despite ~40% decreases in osteocyte volume, mitochondrial volume remained the same at the level of ~20% of cell volume. Mitochondrial membrane potential (MMP) is a critical functional measure of mitochondria. Using tetramethylrhodamine ethyl ester, a potentiometric fluorescent probe, we found ~10% decreases in MMP in osteocytes from GHRKO when compared to osteocytes from control mice, suggesting decreased mitochondrial function with ablation of GHR. To further investigate the changes in mitochondrial function we measured the redox state of the mitochondrial NAD+/NADH pair. We found that GHRKO osteocytes show reduced NADH redox index compared to controls, indicating that the overall levels of NADH in the cell reduced. This suggests reduced TCA cycle activity. The impaired mitochondrial function was further confirmed by assay of cellular respiration. We found that GHRKO osteocytes show reduced oxygen consumption rate as compared to controls, and reduced mitochondrial reserve capacity. This data together with decreased MMP and NADH levels, establish that mitochondrial function is compromised in the absence of GHR in osteocytes

Hepatic osteodystrophy refers to bone loss associated with liver disease. Non-alcoholic fatty liver disease (NAFLD) affects almost 30% of the population and is considered one of the manifestations of the metabolic-syndrome endemic. Clinical studies have demonstrated poor bone health in both children and adults with NAFLD. NAFLD associates with reductions in serum insulin-like growth factor-1 (IGF-1), an established regulator of bone acquisition during growth. Growth hormone (GH) resistance due to liver specific ablation of the GHR (Li-GHRKO), results in NAFLD, systemic dyslipidemia, reductions in serum IGF-1, and severe osteopenia. Since GH tightly regulates hepatic production of IGF-1, target ablation of GHR in liver significantly decreases serum IGF-1 and thus leaving the following questions unanswered: 1) does osteopenia in GH resistance state caused solely by the reductions in liver production of circulating IGF-1 ? And 2) does hepatic GHR regulate bone metabolism via mediators other than IGF-1? To address these questions we have recently created a combined mouse model with liver GHR deletion, in which we restored IGF-1 production via hepatic IGF-1 transgene (HIT), the Li-GHRKO-HIT mouse. We show that normalized serum IGF-1 levels in the Li-GHRKO-HIT mice did NOT resolve NAFLD, or hepatic inflammation. Li-GHRKO-HIT mice showed normal cortical morphology and mechanical strength. However, despite normal serum IGF-1 levels, Li-GHRKO-HIT mice exhibited marked decrease in trabecular bone volume and density associated with increased liver production of tumor necrosis factor a (TNFa), and osteopontin (OPN), an inhibitor of mineral crystal growth. Our data is consistent with a previous report (PMID:23595986), establishing that GH directly inhibits OPN and with several reports showing that GH resistance associates with increased TNFa levels. We conclude that liver GHR activation exerts its action on bone not only via stimulating IGF-1 production in the liver, but also via inhibition of OPN and TNFa, both of which have negative effects on skeletal acquisition

Osteocytes can remove and remodel small amounts of their surrounding bone matrix through osteocytic osteolysis, which results in increased volume occupied by lacunar and canalicular space (LCS). It is well established that cortical bone stiffness and strength are strongly and inversely correlated with vascular porosity, but whether changes in LCS volume caused by osteocytic osteolysis are large enough to affect bone mechanical properties is not known. In the current studies we tested the hypotheses that i) lactation and post-lactation recovery in mice alter the elastic modulus of bone tissue, and ii) such local changes in mechanical properties are related predominantly to alterations in lacunar and canalicular volume rather than bone matrix composition. Mechanical testing was performed using microindentation to measure modulus in regions containing solely osteocytes and no vascular porosity. Lactation caused a significant ( approximately 13%) reduction in bone tissue-level elastic modulus (p < 0.001). After 1 week post-weaning (recovery), bone modulus levels returned to control levels and did not change further after four weeks of recovery. LCS porosity tracked inversely with changes in cortical bone modulus. Lacunar and canalicular void space increased 7% and 15% with lactation, respectively (p < 0.05), then returned to control levels at 1 week after weaning. Neither bone mineralization (assessed by high resolution Backscattered Scanning Electron Microscopy) nor mineral/matrix ratio or crystallinity (assessed by Raman microspectroscopy) changed with lactation. Thus, changes in bone mechanical properties induced by lactation and recovery appear to depend predominantly on changes in osteocyte LCS dimensions. Moreover, this study demonstrates that tissue-level cortical bone mechanical properties are rapidly and reversibly modulated by osteocytes in response to physiological challenge. These data point to a hitherto unappreciated role for osteocytes in modulating and maintaining local bone mechanical properties

Osteocytes can alter their surrounding bone matrix through osteocytic osteolysis, leading to increases in the volume of lacunae and canaliculi. We previously showed that lactation (Lac) caused changes in the tissue-level elastic modulus (Ei) of mouse cortical bone. However, the basis for this change is unclear. Cortical bone modulus is known to depend strongly on vascular porosity, but Lac does not induce intracortical remodeling in mice. Changes in 1) osteocyte lacunar-canalicular space (LCS) and/or 2) composition of bone matrix are hypothesized to account for altered tissue-level mechanical properties in cortical bone. In the current study, we tested whether these parameters could contribute to changes in bone tissue-level mechanical properties seen in Lac and Recovery (Rec). Groups of C57Bl/6 mice (3 mo, n=5/grp) were sacrificed after 2 wks of Lac (Group 1) or underwent forced weaning at 2 wks followed by a further recovery period of 1 wk or 4 wk (Groups 2 and 3). Age-matched controls were also examined. Tissue level elastic modulus (Ei) was measured by microindentation on femoral mid-diaphyseal cross-sections. Lacunar and canalicular areas were measured in images of femoral cross-sections were obtained by super resolution microscopy. Mineralization differences and matrix composition of bone between canaliculi were assessed by back-scattered electron microscopy and Raman microscopy. Ei was reduced by 10-15% after Lac (p<0.005 vs control) but returned to control levels after 1 wk Rec and did not increase further at 4 wks Rec. Under these conditions, lacunar and canalicular areas increased comparably with Lac (19% & 15%, respectively) and retuned to baseline by 1 wk Rec. In contrast, neither mineral distribution in the matrix between canaliculi (Fig 1) nor the composition of that matrix (Fig 2) changed as a result of Lac. These results show that tissue-level cortical bone material properties are rapidly and reversibly modulated during lactation and recovery. As lactation does not cause intracortical remodeling in mice, mechanical changes observed must be driven by osteocytes. Moreover, since lactation did not alter bone matrix mineralization or composition, potential causes of altered microscopic mechanical properties in lactation and recovery were dominated by changes in the LCS void volume resulting from osteocytic osteolysis. These data point to a hitherto unappreciated role for osteocytes in modulating and maintaining local bone material properties