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Jameel Iqbal

Publications and source records attributed to Jameel Iqbal.

At least 19 recordsLinked to original sources
PubMed2006

CD38 is required for priming by TNF-alpha: a mechanism for extracellular coordination of cell fate.

Cytokines are protein messengers that act to modulate the differentiation or activation of their target cells. Bone marrow macrophages can become activated tissue macrophages, dendritic cells, or osteoclasts depending on to which cytokines they are exposed. However, one cytokine can often induce divergent outcomes, suggesting that other signals are needed to establish the specificity of the result. We hypothesize that these signals may derive from the local environment and serve to prime cells to respond toward a specific outcome. Here, it is shown that the cytokine TNF-alpha is capable of affecting the fate of macrophages by upregulating the NADase CD38. CD38 upregulation primes macrophages, such that signals induced by inflammatory stimuli are augmented, while those leading to osteoclast formation are inhibited. We show that TNF-alpha-induced CD38 expression negatively affects the expression of osteoclast markers, while it enhances inflammatory gene expression by decreasing ERK1/2 phosphorylation and increasing NF-kappaB activation. Furthermore, it is shown that CD38 may reduce osteoclastogenesis and increase inflammatory gene induction by decreasing cellular histone deacetylase activity. These results provide a demonstration of how a cytokine can prime cells to differentiate toward a certain lineage or acquire enhanced activation characteristics. Since CD38 is an ectoenzyme, we suggest that the modulation of extracellular NAD(+) metabolism likely serves as a unique mechanism to coordinate the fate of cells within a local environment.

ADP-ribosyl Cyclase 1↗
PubMed2006

Follicle-stimulating hormone stimulates TNF production from immune cells to enhance osteoblast and osteoclast formation.

Declining estrogen production after menopause causes osteoporosis in which the resorption of bone exceeds the increase in bone formation. We recently found that mice deficient in the beta-subunit of follicle-stimulating hormone (FSHbeta) are protected from bone loss despite severe estrogen deficiency. Here we show that FSHbeta-deficient mice have lowered TNFalpha levels. However, TNFalpha-deficient mice are resistant to hypogonadal bone loss despite having elevated FSH, suggesting that TNFalpha is critical to the effect of FSH on bone mass. We find that FSH directly stimulates TNFalpha production from bone marrow granulocytes and macrophages. We also explore how TNFalpha up-regulation induces bone loss. By modeling the known actions of TNFalpha, we attribute the high-turnover bone loss to an expanded osteoclast precursor pool, together with enhanced osteoblast formation. TNFalpha inhibits osteoblastogenesis in the presence of ascorbic acid in culture medium, but in its absence this effect becomes stimulatory; thus, ascorbic acid reverses the true action of TNFalpha. Likewise, ascorbic acid blunts the effects of TNFalpha in stimulating osteoclast formation. We propose that hypogonadal bone loss is caused, at least in part, by enhanced FSH secretion, which in turn increases TNFalpha production to expand the number of bone marrow osteoclast precursors. Ascorbic acid may prevent FSH-induced hypogonadal bone loss by modulating the catabolic actions of TNFalpha.

Animals↗
PubMed2006

Evidence that calcineurin is required for the genesis of bone-resorbing osteoclasts.

Here, we demonstrate that the Ca(2+)/calmodulin-sensitive phosphatase calcineurin is a necessary downstream mediator for osteoclast differentiation. Using quantitative PCR, we detected the calcineurin isoforms Aalpha, Abeta, Agamma (catalytic), and B1 (regulatory) in osteoclast precursor RAW-C3 cells. We found that, although the expression of these isoforms remained relatively unchanged during osteoclast differentiation, there was a profound increase in the expression of their primary substrate for calcineurin, nuclear factor of activated T cells (NFAT)c1. For gain-of-function studies, we incubated osteoclast precursors for 10 min with a calcineurin fusion protein (TAT-calcineurin Aalpha); this resulted in its receptorless influx into >90% of the precursor cells. A marked increase in the expression of the osteoclast differentiation markers tartrate-resistant acid phosphatase (TRAP) and integrin beta(3) followed. In addition, the expression of NFATc1, as well as the alternative substrate for calcineurin, IkappaBalpha, was significantly enhanced. Likewise, transfection with constitutively active NFAT resulted in an increased expression of both TRAP and integrin beta(3). In parallel loss-of-function studies, transfection with dominant-negative NFAT not only inhibited osteoclast formation but also reversed the induction of NFATc1, TRAP, and integrin beta(3) by TAT-calcineurin Aalpha. The expression of these markers was also inhibited by calcineurin Aalpha U1 small nuclear RNA, which significantly reduced calcineurin Aalpha mRNA and protein expression. Consistent with these observations, we observed a reduction in osteoclastogenesis in calcineurin Aalpha(-/-) cells and in osteoclast precursors treated with the calcineurin inhibitors cyclosporin A and FK506. Together, the gain- and loss-of-function experiments establish that calcineurin Aalpha is necessary for osteoclast formation from its precursor and that this occurs via an NFATc1-dependent mechanism.

Acid Phosphatase↗
PubMed2006

Extracellular NAD+ metabolism modulates osteoclastogenesis.

Changes in NAD+ and its metabolites contribute to longevity and age-associated diseases. The role of NAD+ metabolism in bone diseases has however not been investigated, despite the fact that osteoporosis is a leading cause of morbidity in old age. TRAP(+) osteoclast formation from C57 Bl/6J mice was assessed after the addition of varying concentrations of NAD+ metabolites or exogenous ADPribosyl cyclase and NADase enzymes. The NAD+ metabolite cyclic ADPribose (cADPr) or exogenous addition of the enzyme ADPribosyl cyclase stimulated osteoclast formation. Blocking cADPr action with the antagonist 8-Br-cADPr potently inhibited osteoclast formation. In contrast to cADPr, its noncyclized derivative ADPribose (ADPr) or the exogenous addition of NADase both inhibited osteoclastogenesis. As CD38 is the major NAD+ -degrading enzyme present in the bone marrow, these results suggest that CD38-mediated inhibition of osteoclastogenesis is related to its NADase activity, not its ADPribosyl cyclase activity.

ADP-ribosyl Cyclase 1↗
PubMed2006

Selective upregulation of the ADP-ribosyl cyclases CD38 and CD157 by TNF but not by RANK-L reveals differences in downstream signaling.

In macrophages and osteoclast precursors, the cytokines TNF and RANK-L induce similar downstream pathways and share some of the same adaptor molecules. However, despite these similarities, no defined signaling schematic has emerged to show how each cytokine favors particular pathways. In this report, we investigate whether TNF and RANK-L differentially regulate ADP-ribosyl cyclases-enzymes that are unique in being crucial for immunological function yet detrimental to osteoclastogenesis. TNF but not RANK-L led to the sustained upregulation of both CD38 and CD157 as demonstrated by real-time PCR and flow cytometry. Further investigation demonstrated that this upregulation was a result of continuous, direct TNF signaling and involved JNK, and more critically PKC and NF-kappaB. Using this approach allowed us to highlight the relative importance of the PKC, NF-kappaB, and JNK pathways in actualizing proper outcomes of TNF signaling. Albeit speculative, we believe that differences between TNF- and RANK-l-induced activation of downstream signaling pathways, in particular PKC, are crucial for determining whether progenitor cells become geared for immunity or bone resorption.

ADP-ribosyl Cyclase↗
PubMed2006

FSH directly regulates bone mass.

Postmenopausal osteoporosis, a global public health problem, has for decades been attributed solely to declining estrogen levels. Although FSH levels rise sharply in parallel, a direct effect of FSH on the skeleton has never been explored. We show that FSH is required for hypogonadal bone loss. Neither FSHbeta nor FSH receptor (FSHR) null mice have bone loss despite severe hypogonadism. Bone mass is increased and osteoclastic resorption is decreased in haploinsufficient FSHbeta+/- mice with normal ovarian function, suggesting that the skeletal action of FSH is estrogen independent. Osteoclasts and their precursors possess G(i2alpha)-coupled FSHRs that activate MEK/Erk, NF-kappaB, and Akt to result in enhanced osteoclast formation and function. We suggest that high circulating FSH causes hypogonadal bone loss.

Animals↗
PubMed2006

TNF regulates cellular NAD+ metabolism in primary macrophages.

The inflammatory cytokine TNF is known to affect glucose and lipid metabolism, where its action leads to a cachexic state. Despite a well-established connection of TNF to metabolism, the relationship between TNF and NAD(+) metabolism remains unclear. In this report, we evaluated the effects of TNF on NAD(+) metabolism in cells that are TNF's primary autocrine target-macrophages. We designed real-time PCR primers to all NAD(+) metabolic enzymes, which we used to examine TNF-induced changes over time. We found that TNF paradoxically up-regulated enzymes that served to increase NAD(+) levels, such as IDO and PBEF, as well as enzymes that decrease NAD(+) levels, such as CD38 and CD157. The significance of these mRNA changes was evaluated by examining TNF-mediated changes in cellular NAD(+) levels. Treatment of macrophages with TNF decreased NAD(+) levels over time, suggesting that increases in NAD(+)-degrading enzymes were dominant. To evaluate whether this was the case, we measured TNF-mediated changes in NAD(+) levels in animals where CD38 was genetically deleted. In CD38-/- macrophages, the effects of TNF were reversed, with TNF increasing NAD(+) levels over time. The significance of our findings is threefold: (1) we establish that TNF affects NAD(+) metabolism by regulating the expression of major NAD(+) metabolic enzymes, (2) TNF-induced decreases in cellular NAD(+) levels were carried out through the up-regulation of extracellularly situated enzymes, and (3) we provide a mechanism for the observed clinical connection of TNF-dependent diseases to tissue reductions in NAD(+) content.

Animals↗
PubMed2006

Structure and functional regulation of the CD38 promoter.

CD38 has multiple roles in biology, including T lymphocyte signaling, neutrophil migration, neurotransmission, cell proliferation, apoptosis, and bone remodeling. To study the transcriptional control of the CD38 gene, we cloned a putative 1.8 kb promoter fragment from a rabbit genomic DNA library. Primer extension analysis indicated two transcription start sites consistent with the absence of a TATA box. Sequence analysis revealed several AP-1, AP-4, myo-D, GATA, and SP-1 sequences. MC3T3.E1 (osteoblast) or RAW-C3 (osteoclast precursor macrophage) cells were then transfected with the CD38 promoter or its deletion fragments ligated to the luciferase reporter gene. Except for the shortest 41 bp fragment, all fragments showed significant luciferase activity. There was a marked stimulation of basal activity in the 93 bp fragment that contained a GC box and SP-1 site. Furthermore, there were significant differences in the activity of the fragments in MC3T3.E1 and RAW-C3 cells. Intracellular Ca(2+) elevations by ionomycin (10muM) in MC3T3.E1 cells inhibited promoter activity, except in the short 41 bp. In contrast, cAMP elevation by exposure to forskolin (100 microM) inhibited activation of all fragments, except the 0.6 and 1.2kb fragments. Finally, TNF-alpha stimulated promoter activity in RAW-C3 cells transfected with the 93 bp and 1.0 kb fragments, consistent with the stimulation of CD38 mRNA by TNF-alpha. Physiologically, therefore, modulation of the expression of the NAD(+)-sensing enzyme, CD38, by Ca(2+), cAMP, and cytokines, such as TNF-alpha may contribute to coupling the intense metabolic activity of osteoclasts and osteoblasts to their respective bone-resorbing and bone-forming functions.

ADP-ribosyl Cyclase 1↗
PubMed2006

Does TNF have anti-osteoclastogenic actions?

Tumor necrosis factor (TNF) is a pro-resorption agent that leads to bone degradation. Several mechanisms of action have been proposed to account for these effects: TNF directly inhibits osteoblast differentiation; TNF augments osteoclast formation by inducing stromal cells to increase expression of RANKL and macrophage colony-stimulating factor (M-CSF) and decrease that of osteoprotegerin (OPG); and TNF serves to synergize with pathways downstream of RANK to directly increase osteoclast differentiation. All of these actions in sum suggest that TNF dramatically induces osteopenia upon overexpression or injection. However, that the osteopenia seen with TNF is significantly milder than that seen in OPG-/- animals prompted a reevaluation of existing paradigms on TNF action. The hypothesis that TNF directly enhances osteoclast differentiation was tested by examining the effects of TNF on RANKL-induced osteoclast formation and marker expression. The data show that TNF decreased RANKL-induced expression of the osteoclast markers, TRAP and cathepsin K. Furthermore, the addition of 10-60 ng/mL TNF failed to significantly increase RANKL-induced osteoclast differentiation. Instead, data are presented to suggest that the pro-osteoclastogenic actions of TNF are mediated through increases in the number of available osteoclast precursors (macrophages).

Animals↗
PubMed2005

Calcineurin regulates bone formation by the osteoblast.

Two of the most commonly used immunosuppressants, cyclosporine A and tacrolimus (FK506), inhibit the activity of a ubiquitously expressed Ca(2+)/calmodulin-sensitive phosphatase, calcineurin. Because both drugs also cause profound bone loss in humans and in animal models, we explored whether calcineurin played a role in regulating skeletal remodeling. We found that osteoblasts contained mRNA and protein for all isoforms of calcineurin A and B. TAT-assisted transduction of fusion protein TAT-calcineurin Aalpha into osteoblasts resulted in the enhanced expression of the osteoblast differentiation markers Runx-2, alkaline phosphatase, bone sialoprotein, and osteocalcin. This expression was associated with a dramatic enhancement of bone formation in intact calvarial cultures. Calcineurin Aalpha(-/-) mice displayed severe osteoporosis, markedly reduced mineral apposition rates, and attenuated colony formation in 10-day ex vivo stromal cell cultures. The latter was associated with significant reductions in Runx2, bone sialoprotein, and osteocalcin expression, paralleled by similar decreases in response to FK506. Together, the gain- and loss-of-function experiments indicate that calcineurin regulates bone formation through an effect on osteoblast differentiation.

Animals↗
PubMed2005

Molecular regulation of mechanotransduction.

There is a common mechanism for mechanotransduction in cells, regardless of the cell type. Integrins, interacting with their matrix/environment, mediate increases in intracellular Ca2+ levels and activate MAP kinase cascades to cause ERK1/2 phosphorylation. Phosphorylated ERK1/2 causes the activation of the AP-1 family of transcription factors that are necessary for the pro-growth response. The pro-bone growth response involves upregulation of the genes c-fos, IGF-1, cyclooxygenase, and osteocalcin. In osteocytes, increases in intracellular Ca2+ levels may additionally occur by extracellular Ca2+ influx through a stretch-activated ion channel. Each bone cell appears fine-tuned for the type of stimulus, with accessory mechanotransduction signaling pathways, such as calcineurin-mediated activation of the tissue-specific transcription factor NF-AT, adjusting the outcome of signaling in each case.

Animals↗
PubMed2003

TSH is a negative regulator of skeletal remodeling.

The established function of thyroid stimulating hormone (TSH) is to promote thyroid follicle development and hormone secretion. The osteoporosis associated with hyperthyroidism is traditionally viewed as a secondary consequence of altered thyroid function. We provide evidence for direct effects of TSH on both components of skeletal remodeling, osteoblastic bone formation, and osteoclastic bone resorption, mediated via the TSH receptor (TSHR) found on osteoblast and osteoclast precursors. Even a 50% reduction in TSHR expression produces profound osteoporosis (bone loss) together with focal osteosclerosis (localized bone formation). TSH inhibits osteoclast formation and survival by attenuating JNK/c-jun and NFkappaB signaling triggered in response to RANK-L and TNFalpha. TSH also inhibits osteoblast differentiation and type 1 collagen expression in a Runx-2- and osterix-independent manner by downregulating Wnt (LRP-5) and VEGF (Flk) signaling. These studies define a role for TSH as a single molecular switch in the independent control of both bone formation and resorption.

Animals↗
PubMed2003

Disordered osteoclast formation and function in a CD38 (ADP-ribosyl cyclase)-deficient mouse establishes an essential role for CD38 in bone resorption.

We have evaluated the role of the ADP-ribosyl cyclase, CD38, in bone remodeling, a process by which the skeleton is being renewed constantly through the coordinated activity of osteoclasts and osteoblasts. CD38 catalyzes the cyclization of its substrate, NAD+, to the Ca2+-releasing second messenger, cyclic ADP-ribose (cADPr). We have shown previously that CD38 is expressed both in osteoblasts and osteoclasts. Its activation in the osteoclast triggers Ca2+ release through ryanodine receptors (RyRs), stimulation of interleukin-6 (IL-6), and an inhibition of bone resorption. Here, we have examined the consequences of deleting the CD38 gene in mice on skeletal remodeling. We report that CD38-/- mice displayed a markedly reduced bone mineral density (BMD) at the femur, tibia, and lumbar spine at 3 months and at the lumbar spine at 4 months, with full normalization of the BMD at all sites at 5 months. The osteoporosis at 3 months was accompanied by a reduction in primary spongiosa and increased osteoclast surfaces on histomorphometric analysis. Hematopoetic stem cells isolated ex vivo from CD38-/- mice showed a dramatic approximately fourfold increase in osteoclast formation in response to incubation for 6 days with RANK-L and M-CSF. The osteoclasts so formed in these cultures showed a approximately 2.5-fold increase in resorptive activity compared with wild-type cells. However, when adherent bone marrow stromal cells were allowed to mature into alkaline phosphatase-positive colony-forming units (CFU-Fs), those derived from CD38-/- mice showed a significant reduction in differentiation compared with wild-type cells. Real-time RT-PCR on mRNA isolated from osteoclasts at day 6 showed a significant reduction in IL-6 and IL-6 receptor mRNA, together with significant decreases in the expression of all calcineurin A isoforms, alpha, beta, and gamma. These findings establish a critical role for CD38 in osteoclast formation and bone resorption. We speculate that CD38 functions as a cellular NAD+ "sensor," particularly during periods of active motility and secretion.

ADP-ribosyl Cyclase↗
PubMed2003

Impaired osteoblastic differentiation, reduced bone formation, and severe osteoporosis in noggin-overexpressing mice.

We describe the effects of the overexpression of noggin, a bone morphogenetic protein (BMP) inhibitor, on osteoblast differentiation and bone formation. Cells of the osteoblast and chondrocyte lineages, as well as bone marrow macrophages, showed intense beta-gal histo- or cytostaining in adult noggin+/- mice that had a LacZ transgene inserted at the site of noggin deletion. Despite identical BMP levels, however, osteoblasts of 20-month-old C57BL/6J and 4-month-old senescence-accelerated mice (SAM-P6 mice) had noggin expression levels that were approximately fourfold higher than those of 4-month-old C57BL/6J and SAM-R1 (control) mice, respectively. U-33 preosteoblastic cells overexpressing the noggin gene showed defective maturation and, in parallel, a decreased expression of Runx-2, bone sialoprotein, osteocalcin, and RANK-L. Noggin did not inhibit the ligandless signaling and pro-differentiation action of the constitutively activated BMP receptor type 1A, ca-ALK-3. Transgenic mice overexpressing noggin in mature osteocalcin-positive osteoblasts showed dramatic decreases in bone mineral density and bone formation rates with histological evidence of decreased trabecular bone and CFU-osteoblast colonies at 4 and 8 months. Together, the results provide compelling evidence that noggin, expressed in mature osteoblasts, inhibits osteoblast differentiation and bone formation. Thus, the overproduction of noggin during biological aging may result in impaired osteoblast formation and function and hence, net bone loss.

Aging↗
PubMed2003

Cinacalcet hydrochloride (Amgen).

Amgen Inc and Kirin Brewery Co Ltd, under license from NPS Pharmaceuticals Inc, are developing cinacalcet hydrochloride, the lead compound in a series of calcimimetics, for the potential treatment of primary and secondary hyperparathyroidism. Tecalcet hydrochloride was the first compound from this class to be extensively studied and most of the pharmacological data disclosed is for this compound. Cinacalcet hydrochloride was developed in an effort to improve on the poor pharmacokinetics and metabolism of tecalcet hydrochloride.

Animals↗
PubMed2002

Molecular cloning, expression, and function of osteoclastic calcineurin Aalpha.

This study explores the role of the calmodulin- and Ca(2+)-sensitive phosphatase calcineurin A in the control of bone resorption by mature osteoclasts. We first cloned full-length calcineurin Aalpha and Abeta cDNA from a rabbit osteoclast library. Sequence analysis revealed an approximately 95 and 86% homology between the amino acid and the nucleotide sequences, respectively, of the two isoforms. The two rabbit isoforms also showed significant homology with the mouse, rat, and human homologs. In situ RT-PCR showed evidence of high levels of expression of calcineurin Aalpha mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin Aalpha expression in cells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 x 10(-7) M) and FK506 (5 x 10(-9) and 5 x 10(-7) M). We then constructed a fusion protein comprising calcineurin Aalpha and TAT, a 12-amino acid-long arginine-rich sequence of the human immunodeficiency virus protein. Others have previously shown that the fusion of proteins to this sequence results in their receptor-less transduction into cells, including osteoclasts. Similarly, unfolding of the TAT-calcineurin Aalpha fusion protein by shocking with 8 M urea resulted in its rapid influx, within minutes, into as many as 90% of all freshly isolated rat osteoclasts, as was evident on double immunostaining with anti-calcineurin Aalpha and anti-TAT antibodies. Pit assays performed with TAT-calcineurin Aalpha-positive osteoclasts revealed a concentration-dependent (10-200 nM) attenuation of bone resorption in the absence of cell cytotoxicity or changes in cell number. TAT-hemaglutinin did not produce significant effects on bone resorption or cell number. The study suggests the following: 1) the 61-kDa protein phosphatase calcineurin Aalpha can be effectively tranduced into osteoclasts by using the TAT-based approach, and 2) the transduced protein retains its capacity to inhibit osteoclastic bone resorption.

Amino Acid Sequence↗
PubMed2002

A novel mechanism for coupling cellular intermediary metabolism to cytosolic Ca2+ signaling via CD38/ADP-ribosyl cyclase, a putative intracellular NAD+ sensor.

CD38 is an ectocyclase that converts NAD+ to the Ca2+-releasing second messenger cyclic ADP-ribose (cADPr). Here we report that in addition to CD38 ecto-catalysis, intracellularly expressed CD38 may catalyze NAD+-->cADPr conversion to cause cytosolic Ca2+ release. High levels of CD38 were found in the plasma membranes, endoplasmic reticulum, and nuclear membranes of osteoblastic MC3T3-E1 cells. More important, intracellular CD38 was colocalized with target ryanodine receptors. The cyclase also converted a NAD+ surrogate, NGD+, to its fluorescent product, cGDPr (Km approximately 5.13 microM). NAD+ also triggered a cytosolic Ca2+ signal. Similar results were obtained with NIH3T3 cells, which overexpressed a CD38-EGFP fusion protein. The Delta(-49)-CD38-EGFP mutant with a deleted amino-terminal tail and transmembrane domain appeared mainly in the mitochondria with an expected loss of its membrane localization, but the NAD+-induced cytosolic Ca2+ signal was preserved. Likewise, Ca2+ release persisted in cells transfected with the Myr-Delta(-49)-CD38-EGFP or Delta(-49)-CD38-EGFP-Fan mutants, both directed to the plasma membrane but in an opposite topology to the full-length CD38-EGFP. Finally, ryanodine inhibited Ca2+ signaling, indicating the downstream activation of ryanodine receptors by cADPr. We conclude that intracellularly expressed CD38 might link cellular NAD+ production to cytosolic Ca2+ signaling.

3T3 Cells↗