Linda R. Halstead

Altered mineral metabolism in glucocorticoid-induced osteopenia. Effect of 25-hydroxyvitamin D administration.

Parameters of mineral and bone metabolism were studied in 17 patients treated chronically with supraphysiologic doses of glucocorticoids. When compared to 15 matched normal subjects, the patient group exhibited similar serum 25-hydroxyvitamin D (25-OHD) levels, decreased intestinal 47Ca absorption, increased serum immunoreactive parathyroid hormone, and decreased forearm bone mass. Iliac crest bone biopsies revealed a decreased bone formation rate and increased osteoclast number. Treatment with 25-OHD (mean dose 4.03 micrograms/d) and calcium (500 mg/d) in nine patients produced a 46% increase in 47Ca absorption (P less than 0.001) and a 54% decrease in serum immunoreactive parathyroid hormone (P less than 0.001) by 3 mo. In addition, by 12 mo the treatment group exhibited (a) a 13.2 +/- 5.1% increase in metaphyseal (P less than 0.001) and a 2.1 +/- 0.4% increase in diaphyseal (P less than 0.05) forearm bone mass, and (b) significant decreases in cortical and endosteal osteoclast number. Biochemical and bone mass changes persisted through 18 mo. No significant changes in any parameter occurred in eight control patients administered calcium 100 mg/d. It is concluded that treatment with 25-OHD and calcium can significantly improve parameters of mineral and bone metabolism in patients with glucocorticoid-induced osteopenia.

Erk is essential for growth, differentiation, integrin expression,and cell function in human osteoblastic cells

Extracellular signal-regulated kinases (Erks), members of the mitogen-activated protein kinase superfamily, play an important role in cell proliferation and differentiation. In this study we employed a dominant negative approach to determine the role of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped retrovirus encoding either a mutated Erk1 protein with a dominant negative action against both Erk1 and Erk2 (Erk1DN cells) or the LacZ protein (LacZ cells) as a control. Both basal and growth factor-stimulated MAPK activity and cell proliferation were inhibited in Erk1DN cells. Expression of Erk1DN protein suppressed both osteoblast differentiation and matrix mineralization by decreasing alkaline phosphatase activity and the deposition of bone matrix proteins. Cell adhesion to collagen, osteopontin, and vitronectin was decreased in Erk1DN cells as compared with LacZ cells. Cell spreading and migration on these matrices were also inhibited. In Erk1DN cells, expression of αβ1, αvβ3, and αvβ5 integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these integrins was inhibited in Erk1DN cells. These data suggest that Erks are not only essential for the growth and differentiation of osteoblasts but also are important for osteoblast adhesion, spreading, migration, and integrin expression.

Interaction of Diphenylhydantoin (Phenytoin) and Phenobarbital with Hormonal Mediation of Fetal Rat Bone Resorption In Vitro

Chronic administration of high doses of anticonvulsant drugs frequently produces classic osteomalacia with bone histologic changes characteristic of increased parathyroid hormone (PTH) effect in man. However, several reports have documented defects in calcified tissue metabolism suggestive of an end-organ resistance to PTH after chronic anticonvulsant drug therapy. To examine the direct action of anticonvulsant drugs on bone resorption, we investigated the effects of diphenylhydantoin (phenytoin) (DPH) (100-200 mug/ml) and phenobarbital (10-400 mug/ml) on basal and hormonally mediated resorption 5-day cultures of fetal rat forelimb rudiments. In this system both drugs significantly inhibited basal and PTH-stimulated (45)Ca and [(3)H]hydroxyproline release, as well as 1,25-dihydroxyvitamin D(3)-stimulated (45)Ca release. The effects of DPH and phenobarbital were additive, with DPH exhibiting a several-fold more potent inhibitory effect than phenobarbital. Whereas DPH exhibited a striking synergism with the inhibitory effects of human calcitonin (HCT) on PTH-induced resorption, the effect of phenobarbital was merely additive to that of HCT. PTH and PTH plus HCT-induced increases in bone cyclic AMP (cAMP) content were significantly inhibited by DPH but not by phenobarbital. However, in contrast to effects on (45)Ca release, DPH inhibition of cAMP generation was not accentuated in the presence of HCT. It is concluded that: (a) both DPH and phenobarbital can directly inhibit basal and hormonally stimulated bone resorption, with DPH being much more potent in this regard; (b) DPH appears to inhibit bone resorption via a cAMP-independent mechanism and has an additional suppressive effect on PTH-induced cAMP generation; and (c) the synergistic interaction of DPH and HCT in inhibiting (45)Ca release occurs at a site independent of cAMP generation.

Human osteoblasts in vitro secrete tissue inhibitor of metalloproteinases and gelatinase but not interstitial collagenase as major cellular products.

Human osteoblast cultures (hOB) were examined for the production of interstitial collagenase, tissue inhibitor of metalloproteinases (TIMP), and gelatinolytic enzymes. Cells were isolated by bacterial collagenase digestion of trabecular bone (vertebra, rib, tibia, and femur) from 11 subjects (neonatal to adult). Confluent cultures were exposed to phorbol 12-myristate 13-acetate, PTH, PGE2, epidermal growth factor, 1,25(OH)2 vitamin D3, recombinant human IL-1 beta, and dexamethasone. Collagenase and TIMP were assayed immunologically and also by measurements of functional activity. Collagenase was not secreted in significant quantities by human bone cells under any tested condition. Furthermore, collagenase mRNA could not be detected in hOB. However, hOB spontaneously secreted large amounts of TIMP for at least 72 h in culture. hOB TIMP was found to be identical to human fibroblast TIMP by double immunodiffusion, metabolic labeling and immunoprecipitation, Northern blot analysis, and stoichiometry of collagenase inhibition. SDS-substrate gel electrophoresis of hOB-conditioned media revealed a prominent band of gelatinolytic activity at 68 kD, and specific polyclonal antisera established its identity with the major gelatinolytic protease of human fibroblasts. Abundant secretion of gelatinolytic, but not collagenolytic, enzymes by hOB may indicate that human osteoblasts do not initiate and direct the cleavage of osteoid collagen on the bone surface, but may participate in the preparation of the bone surface for osteoclast attachment by removal of denatured collagen peptides. The constitutive secretion of TIMP may function to regulate metalloproteinase activity.

Activation of Vascular Smooth Muscle Parathyroid Hormone Receptor Inhibits Wnt/β-Catenin Signaling and Aortic Fibrosis in Diabetic Arteriosclerosis

Rationale: Vascular fibrosis and calcification contribute to diabetic arteriosclerosis, impairing Windkessel physiology necessary for distal tissue perfusion. Wnt family members, upregulated in arteries by the low-grade inflammation of “diabesity,” stimulate type I collagen expression and osteogenic mineralization of mesenchymal progenitors via &bgr;-catenin. Conversely, parathyroid hormone (PTH) inhibits aortic calcification in low-density lipoprotein receptor (LDLR)-deficient mice fed high fat diabetogenic diets (HFD). Objective: We sought to determine the impact of vascular PTH receptor (PTH1R) activity on arteriosclerotic Wnt/&bgr;-catenin signaling in vitro and in vivo. We generated SM-caPTH1R transgenic mice, a model in which the constitutively active PTH1R variant H223R (caPTH1R) is expressed only in the vasculature. Methods and Results: The caPTH1R inhibited Wnt/&bgr;-catenin signaling, collagen production, and vascular smooth muscle cell proliferation and calcification in vitro. Transgenic SM-caPTH1R;LDLR+/− mice fed HFD develop diabesity, with no improvements in fasting serum glucose, cholesterol, weight, body composition, or bone mass versus LDLR+/− siblings. SM-caPTH1R downregulated aortic Col1A1, Runx2, and Nox1 expression without altering TNF, Msx2, Wnt7a/b, or Nox4. The SM-caPTH1R transgene decreased aortic &bgr;-catenin protein accumulation and signaling in diabetic LDLR+/− mice. Levels of aortic superoxide (a precursor of peroxide that activates pro–matrix metalloproteinase 9 and osteogenic signaling in vascular smooth muscle cells) were suppressed by the SM-caPTH1R transgene. Aortic calcification, collagen accumulation, and wall thickness were concomitantly reduced, enhancing vessel distensibility. Conclusions: Cell-autonomous vascular smooth muscle cell PTH1R activity inhibits arteriosclerotic Wnt/&bgr;-catenin signaling and reduces vascular oxidative stress, thus limiting aortic type I collagen and calcium accrual in diabetic LDLR-deficient mice.

Anticonvulsant drug-induced osteomalacia: Alterations in mineral metabolism and response to vitamin D3 administration

SummaryParameters of mineral metabolism were examined in 6 patients with moderately severe anticonvulsant drug-induced osteomalacia. Compared to 15 matched controls, the patients exhibited significantly reduced serum calcium, inorganic phosphate, and 25-hydroxyvitamin D concentration, elevated serum alkaline phosphatase and immunoreactive parathyroid hormone (iPTH) concentration, reduced intestinal47Ca absorption, reduced urinary calcium and increased urinary hydroxyproline excretion, and reduced forearm bone mass. Intestinal absorption of vitamin D3 was normal. Following 4 months of treatment with vitamin D3 (4000 units/day), serum 25-OHD concentration was increased to 3 times mean normal values and all parameters except serum iPTH, urinary calcium excretion, and forearm bone mass were returned to levels not significantly different from normal. Serum iPTH concentration was reduced by 39% (P<0.05); 24-h urinary calcium excretion rose by 98% (P<0.001), and forearm bone mass increased by 5.6% (P<0.05). It is concluded that moderate-dose vitamin D3 supplementation is effective in normalizing parameters of mineral metabolism in this disorder, despite evidence of resistance to the biologic effects of vitamin D.

Effect of CYP1A1 Gene Polymorphisms on Estrogen Metabolism and Bone Density

In this study, we evaluated the effect of polymorphisms of the CYP1A1 gene, linked to hormone‐related cancers, on estrogen metabolism and BMD. We found that variants carrying the A allele (CA and AA) for the C4887A polymorphism have a significantly higher degree of estrogen catabolism and lower femoral BMD.

Comparison of subacute effects of oxazacort and prednisone on mineral metabolism in man

SummaryProlonged therapeutic administration of prednisone or other corticosteroids frequently produces severe osteopenia with an increased incidence of bone fractures. Recent efforts to decrease the severity of corticosteroid-induced osteopenia have included the development of corticosteroid analogues designed to possess diminished bone-wasting effects relative to their anti-inflammatory activity. We compared the effects of an oxazoline derivative of prednisolone, oxazacort (azacortinol), with those of prednisone on mineral metabolism in man. After a 12-day equilibration period on a 600 mg/day calcium diet, normal volunteers were studied for 15 days during treatment with either prednisone (20 mg/day, 12 subjects) or oxazacort (25 mg/day, 10 subjects). There was no difference between the two groups with regard to the effects of each corticosteroid on serum ionized calcium, phosphate, alkaline phosphatase, immunoreactive parathyroid hormone (iPTH), and 25-hydroxyvitamin D (25OHD) concentrations. Both corticosteroids suppressed intestinal47Ca absorption to a similar degree after 15 days of treatment (prednisone: −28.5±7.5, oxazacort: −30.2±4.4% of initial values). Although both corticosteroids increased 24-h urinary calcium excretion significantly above pretreatment values, this effect was less marked in the oxazacort-treated subjects. The mean cumulative 15-day increase in urinary calcium excretion in the prednisone-treated group (+326 ± 54 mg/g creatinine/24 h) was more than twice as great as that in the oxazacort-treated group (+146 ± 48 mg/g creatinine/24 h), a difference significant atP<0.001. It is concluded that the increase in urinary calcium excretion, and presumably the negative calcium balance, produced by a 2-week administration of oxazacort is significantly less pronounced than that produced by an equivalent dose of prednisone.

An Intact N Terminus Is Required for the Anabolic Action of Parathyroid Hormone on Adult Female Rats

Intermittent administration of parathyroid hormone (PTH) peptides increases bone density in animal and human models of osteoporosis. In vitro studies have demonstrated that PTH analogs lacking the first two amino acids can stimulate cell proliferation in certain cell systems, whereas fragments with an intact N terminus can be antimitogenic. We have tested whether the truncated PTH(3–38) fragment may be a better “anabolic analog” than PTH(1–38) by monitoring bone density and biomechanical properties of the femur in 6‐month‐old ovariectomized (OVX) rats. Either PTH fragment was administered subcutaneously (8 μg/100 g of body weight) 5 days/week, for 4 weeks, starting 1 week after surgery. During the entire study, untreated OVX rats lost 12.1 ± 4.4% of their initial bone density. PTH(1–38) reversed the initial bone loss, leading to complete restoration of presurgery values after 4 weeks of treatment. Conversely, administration of PTH(3–38) resulted in 13.2 ± 5.8% bone loss, while continuous estrogen infusion (10 μg/kg/day) prevented bone loss but did not reverse it. Sham‐operated animals also experienced significant bone loss in the vehicle and PTH(3–38)–treated groups (−4.5 ± 6.7%, and −7.6 ± 2.8%, respectively), whereas a significant gain in bone density (+4.4 ± 5.6%) was observed in the rats treated with PTH(1–38). A bone quality factor (index of strain energy loss) and the impact strength (resistance to fracture) were 25% and 44% lower in femurs explanted from OVX a0nimals treated with either vehicle or PTH(3–38), compared with sham‐operated animals. On the contrary, no difference was observed between OVX and control animals after treatment with PTH(1–38), indicating a preservation of the capacity to withstand mechanical stress. Thus, PTH(1–38) counteracts estrogen‐dependent loss of mineral density and bone biomechanical properties and increases bone density in estrogen‐replete animals. An intact N terminus sequence is necessary for this anabolic action of PTH.

Four and half lim protein 2 (FHL2) stimulates osteoblast differentiation

FHL2, a molecule that interacts with many integrins and transcription factors, was found to play an important role in osteoblast differentiation. Overexpression of FHL2 increases the accumulation of osteoblast differentiation markers and matrix mineralization, whereas FHL2 deficiency results in inhibition of osteoblast differentiation and decreased bone formation.