# Is PPARγ a Prospective Player in HIV-1-Associated Bone Disease?

Authors: Eoin J. Cotter; Patrick W. Mallon; Peter P. Doran
Journal: PPAR Research
 (2009)

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## Abstract

Currently infection with the human immunodeficiency virus-1 (HIV-1) is in most instances a chronic disease that can be controlled by effective antiretroviral therapy (ART). However, chronic use of ART has been associated with a number of toxicities; including significant reductions in bone mineral density (BMD) and disorders of the fat metabolism. The peroxisome proliferator-activated receptor gamma (PPARγ) transcription factor is vital for the development and maintenance of mature and developing adipocytes. Alterations in PPARγ expression have been implicated as a factor in the mechanism of HIV-1-associated lipodystrophy. Both reduced BMD and lipodystrophy have been well described as complications of HIV-1 infection and treatment, and a question remains as to their interdependence. Interestingly, both adipocytes and osteoblasts are derived from a common precursor cell type; the mesenchymal stem cell. The possibility that dysregulation of PPARγ (and the subsequent effect on both osteoblastogenesis and adipogenesis) is a contributory factor in the lipid- and bone-abnormalities observed in HIV-1 infection and treatment has also been investigated. This review deals with the hypothesis that dysregulation of PPARγ may underpin the bone abnormalities associated with HIV-1 infection, and treats the current knowledge and prospective developments, in our understanding of PPARγ involvement in HIV-1-associated bone disease.


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## Body

## 1. Introduction

Aside from the serious effects on the cells of the immune system, HIV-1 infection  and its treatment have been
associated with disorders in other tissues, most notably bone [1, 2] and adipose [3–6] tissues,  where reduced bone mineral density (BMD) and abnormalities of the lipid metabolism (lipodystrophy,  dyslipidemia,
and insulin resistance) have been described. In both disorders (particularly
those of the adipose tissue), antiretroviral treatment is believed to play a
major role, but the contribution of underlying HIV-1 infection has yet to be
elucidated, and therefore cannot be ignored as a potential causative factor.PPARγ is a nuclear membrane bound transcription factor which regulates a
number of genes involved in adipogenesis from common precursor cells type
(mesenchymal stem cells), maturation of preadipocytes, lipid accumulation,
and maintenance of adipogenic phenotype [7, 8]. As such, it is not surprising
that a number of recent studies have indicated that certain drugs known to be associated
with lipodystrophy dysregulate PPARγ
[9, 10]. The
involvement of PPARγ in HIV-1-associated bone disease is
an area that has been little studied to date; however numerous studies suggest
that PPARγ plays a role in conditions such as
osteoporosis in the absence of HIV-1 or ART, and increased adipocyte content of
osteoporotic bone has been reported [10–12]. In addition,
osteoblasts—the cells
responsible for depositing bone—are derived from mesenchymal
stem cells, and evidence suggests that the balance of PPARγ  and the pro-osteogenic runt-related transcription
factor-2 (RUNX-2) is a key in the determination of mesenchymal stem cell fate
[13–15]  (see Figure 1). This review will introduce the current knowledge of the role of PPARγ in
bone biology in normal and disease states, and discuss its potential as a
mechanism for HIV-1-associated bone disease.Figure 1
Factors governing normal osteogenesis and
adipogenesis  from mesenchymal stem cells. Multipotent mesenchymal
stem cells can differentiate into a number of cell types, including adipocytes
and osteoblasts. (⊥ indicates
inhibition; ↓ indicates stimulation). The
transcriptional coactivator Taz negatively regulates adipogenesis and promotes
osteogenesis through suppression of PPARγ and
activation of RUNX-2, while overexpression of PPARγ can reduce bone formation. Also, a number
of other factors such as secreted proteins from Wnt family promote the differentiation and maintenance
of osteoblasts while reducing the differentiation of the adipocytes. In
addition, factors secreted by mature adipocytes, such as leptin and estrogen, can
increase bone mass in vivo.


## 2. HIV-1-Associated Bone Disease

Osteoporosis is defined as a
reduction in the bone mass and disruption of the microarchitecture of the bone
which leads to a greatly increased risk of fractures, while osteopenia is a
lesser reduction in bone density and strength which may remain asymptomatic,
but can precede actual osteoporosis. The world health organization (WHO) definitions
specify t-scores between −1 and −2.5 as being indicative of osteopenia, while t-scores of less than −2.5
are indicative of osteoporosis [16]. Fractures resulting from osteoporosis
affect one in two women and one in five men over the age of 50, and are a
significant financial burden to health services, with an estimated combined
annual cost of 30 billion Euro in the EU [17].As will be discussed further, bone remodeling is
dependent on the opposing functions of two cell types, osteoblasts, which make
new bone (bone formation), and osteoclasts, which destroy old bone (bone
resorption). Therefore, the balance between the number and activity of
osteoclasts and osteoblasts is crucial in normal bone homeostasis; the
perturbation of which can directly lead to increased bone fragility and fracture
risk. Two important molecules: macrophage colony-stimulating factor (M-CSF) and
receptor for activation of nuclear factor-kappa B ligand (RANKL) produced from
osteoblast/stromal cells regulate the differentiation, function, and survival
of osteoclasts, while the transcription factors, RUNX-2 and Osterix, have been
reported to regulate osteoblast differentiation [18].


### 2.1. HIV-1 Infection and Bone Disease

Bone metabolism
in HIV-infected individuals has been studied since the late 1980s, although the
number of early studies is
somewhat limited. Before the widespread use of highly active ART, studies
indicated that bone mineral metabolism was only minimally affected in
HIV-infected patients. Serrano et al. assessed histomorphometry in HIV-positive patients and found that parameters of
histomorphometry such as serum osteocalcin were found to be lower in patients who, according
to the Centers for Disease Control (CDC) classification, had greater disease
severity [19]. Paton et al. reported that 45 HIV-infected patients had marginally lower BMD at the lumbar
spine. None of the patients had reduced BMD to levels associated with a
diagnosis of osteoporosis [20]. More recently however, it
became clear that reduced BMD is also frequent in the absence of therapy [21–24]. In a study
by McGowan et al., the
prevalence of osteopenia among antiretroviral-naive HIV-positive individuals to be approximately 28%, which is approximately 50% greater than the expected incidence in the general, uninfected population [25]. Studies which have included patients with more advanced HIV disease who have received
treatment for longer periods have reported prevalence of 40% to 50% [26, 27], placing
reduced BMD among the most common HIV-1-associated metabolic toxicities.
Amiel et al. also assessed BMD
in 48 HIV-infected treatment-naive patients, 49 HIV-infected patients on
protein inhibitors, 51 HIV-infected patients on no-protein inhibitors, and 81
HIV-uninfected control subjects. The results showed a significant decrease of
BMD (9%) in all HIV-infected patients compared to the control subjects,
occurring concurrently with a lower bone alkaline phosphatase and higher urinary cross-laps/Cr. [28]. The clinical impact of this
reduced BMD is beginning to be examined; recent studies in a large American
health care system, involving 8526 HIV infected patients and over 2 million
control subjects, demonstrated that the prevalence of any fracture type was significantly higher in the HIV-infected population (2.87 versus 1.77 fractures/100 persons, P=.002).
This study did not specify the treatment status of their subjects, but the data
suggests that HIV-1-related
fractures are a significant and growing clinical issue [29].


### 2.2. Antiviral Treatment and Bone Diseases

Antiretroviral
treatment (ART) is a complex therapeutic regimen, in which patients typically
take 2-3 agents selected from an array of 30 approved antiretroviral agents.
ART, in general, comprises of two major therapeutic strategies: a protease
inhibitor- (PI-) based regimen and a nucleoside reverse transcriptase inhibitor-
(NRTI-) based regimen. The PI-based regimen uses one or two PIs combined with
two NRTIs, whereas the NRTI-based regimen uses two NRTIs combined with one non-nucleoside
reverse transcriptase inhibitor (NNRTI). With more effective therapies as a
result of HAART, the prevalence of HAART-associated bone diseases has increased
[30].A higher incidence of reduced BMD has been
clinically associated with both PI and NRTI uses. Tebas et al. determined that
in HIV-1 patients receiving PIs about 50% of the patients
had osteopenia and other 21% had osteoporosis [31]. This incidence is
significantly increased compared to patients without therapy or normal
controls. Studies by Moore et al.
confirmed that 71% of HIV-infected patients on PI therapy have reduced BMD [32].
Similarly, Carr et al. reported
that 3% of 44 HIV-infected patients receiving NRTIs developed osteoporosis and
22% developed osteopenia [33], while in a study examining HIV-1-infected men
Mallon determined a reduction in BMD beginning at 48 weeks postinitiation of
treatment [6]. Tsekes et al.
determined BMD and whole body fat by dual energy X-ray absorbance (DEXA) of HIV-infected patients
receiving zidovudine and other NRTIs and found significant decreases in both
body fat and BMD [34]. In addition, the recent analysis by Brown and Qaqish [35]
also reported 2.5-fold increased odds of reduced BMD in ART-treated patients
compared with ART-naive patients (95% CI 1.8, 3.7). However, most studies are in agreement that
traditional risk factors for osteoporosis, such as ethnic variations, female
sex, increasing age, low body mass index, and time since menopause, are all
independent predictors of osteopenia/osteoporosis [36–40].In addition, it has been noted
that HIV-infected patients have an increased risk for osteonecrosis of the hip
[41]. Keruly et al. reported 15
cases of avascular hip necrosis in HIV-infected patients and suggested that the
incidence of osteonecrosis in HIV-infected patients was higher than the general
HIV-negative population [42]. It is not known whether this phenomenon is
attributable to HIV-1 infection itself, HAART, or other HIV-associated
complications.The mechanisms by which either
HIV-1 or its treatment induces
reduced BMD are as yet unclear, and several researchers have suggested that
reduced vitamin D levels observed in HIV-1-infected patients, and particularly the reduced
levels of the biologically active metabolite 1,25(OH)2D (which is
the natural ligand for the vitamin D receptor (VDR)), may contribute to reduced
BMD [43]. Studies have demonstrated that the level of 1,25(OH)2D in
HIV-1-infected patients is
between 5 and 50% lower than
that in infected patients [24, 44, 45].  In addition, studies have indicated that
patients receiving treatment are more likely to have greater reductions in
1,25(OH)2D, with a recent Dutch study suggesting that NNRTI
treatment may increase the risk of vitamin D deficiency [46, 47]. In addition,
the latter study demonstrated that patients receiving treatment also have
increased parathyroid hormone (PTH) levels, increasing the potential risk of reduced
bone mass.In short, HIV-1-associated bone
disorders are a significant and increasingly well-defined clinical issue.
However, the molecular basis underpinning these clinical observations remains
to be fully explained.


## 2.1. HIV-1 Infection and Bone Disease

Bone metabolism
in HIV-infected individuals has been studied since the late 1980s, although the
number of early studies is
somewhat limited. Before the widespread use of highly active ART, studies
indicated that bone mineral metabolism was only minimally affected in
HIV-infected patients. Serrano et al. assessed histomorphometry in HIV-positive patients and found that parameters of
histomorphometry such as serum osteocalcin were found to be lower in patients who, according
to the Centers for Disease Control (CDC) classification, had greater disease
severity [19]. Paton et al. reported that 45 HIV-infected patients had marginally lower BMD at the lumbar
spine. None of the patients had reduced BMD to levels associated with a
diagnosis of osteoporosis [20]. More recently however, it
became clear that reduced BMD is also frequent in the absence of therapy [21–24]. In a study
by McGowan et al., the
prevalence of osteopenia among antiretroviral-naive HIV-positive individuals to be approximately 28%, which is approximately 50% greater than the expected incidence in the general, uninfected population [25]. Studies which have included patients with more advanced HIV disease who have received
treatment for longer periods have reported prevalence of 40% to 50% [26, 27], placing
reduced BMD among the most common HIV-1-associated metabolic toxicities.
Amiel et al. also assessed BMD
in 48 HIV-infected treatment-naive patients, 49 HIV-infected patients on
protein inhibitors, 51 HIV-infected patients on no-protein inhibitors, and 81
HIV-uninfected control subjects. The results showed a significant decrease of
BMD (9%) in all HIV-infected patients compared to the control subjects,
occurring concurrently with a lower bone alkaline phosphatase and higher urinary cross-laps/Cr. [28]. The clinical impact of this
reduced BMD is beginning to be examined; recent studies in a large American
health care system, involving 8526 HIV infected patients and over 2 million
control subjects, demonstrated that the prevalence of any fracture type was significantly higher in the HIV-infected population (2.87 versus 1.77 fractures/100 persons, P=.002).
This study did not specify the treatment status of their subjects, but the data
suggests that HIV-1-related
fractures are a significant and growing clinical issue [29].


## 2.2. Antiviral Treatment and Bone Diseases

Antiretroviral
treatment (ART) is a complex therapeutic regimen, in which patients typically
take 2-3 agents selected from an array of 30 approved antiretroviral agents.
ART, in general, comprises of two major therapeutic strategies: a protease
inhibitor- (PI-) based regimen and a nucleoside reverse transcriptase inhibitor-
(NRTI-) based regimen. The PI-based regimen uses one or two PIs combined with
two NRTIs, whereas the NRTI-based regimen uses two NRTIs combined with one non-nucleoside
reverse transcriptase inhibitor (NNRTI). With more effective therapies as a
result of HAART, the prevalence of HAART-associated bone diseases has increased
[30].A higher incidence of reduced BMD has been
clinically associated with both PI and NRTI uses. Tebas et al. determined that
in HIV-1 patients receiving PIs about 50% of the patients
had osteopenia and other 21% had osteoporosis [31]. This incidence is
significantly increased compared to patients without therapy or normal
controls. Studies by Moore et al.
confirmed that 71% of HIV-infected patients on PI therapy have reduced BMD [32].
Similarly, Carr et al. reported
that 3% of 44 HIV-infected patients receiving NRTIs developed osteoporosis and
22% developed osteopenia [33], while in a study examining HIV-1-infected men
Mallon determined a reduction in BMD beginning at 48 weeks postinitiation of
treatment [6]. Tsekes et al.
determined BMD and whole body fat by dual energy X-ray absorbance (DEXA) of HIV-infected patients
receiving zidovudine and other NRTIs and found significant decreases in both
body fat and BMD [34]. In addition, the recent analysis by Brown and Qaqish [35]
also reported 2.5-fold increased odds of reduced BMD in ART-treated patients
compared with ART-naive patients (95% CI 1.8, 3.7). However, most studies are in agreement that
traditional risk factors for osteoporosis, such as ethnic variations, female
sex, increasing age, low body mass index, and time since menopause, are all
independent predictors of osteopenia/osteoporosis [36–40].In addition, it has been noted
that HIV-infected patients have an increased risk for osteonecrosis of the hip
[41]. Keruly et al. reported 15
cases of avascular hip necrosis in HIV-infected patients and suggested that the
incidence of osteonecrosis in HIV-infected patients was higher than the general
HIV-negative population [42]. It is not known whether this phenomenon is
attributable to HIV-1 infection itself, HAART, or other HIV-associated
complications.The mechanisms by which either
HIV-1 or its treatment induces
reduced BMD are as yet unclear, and several researchers have suggested that
reduced vitamin D levels observed in HIV-1-infected patients, and particularly the reduced
levels of the biologically active metabolite 1,25(OH)2D (which is
the natural ligand for the vitamin D receptor (VDR)), may contribute to reduced
BMD [43]. Studies have demonstrated that the level of 1,25(OH)2D in
HIV-1-infected patients is
between 5 and 50% lower than
that in infected patients [24, 44, 45].  In addition, studies have indicated that
patients receiving treatment are more likely to have greater reductions in
1,25(OH)2D, with a recent Dutch study suggesting that NNRTI
treatment may increase the risk of vitamin D deficiency [46, 47]. In addition,
the latter study demonstrated that patients receiving treatment also have
increased parathyroid hormone (PTH) levels, increasing the potential risk of reduced
bone mass.In short, HIV-1-associated bone
disorders are a significant and increasingly well-defined clinical issue.
However, the molecular basis underpinning these clinical observations remains
to be fully explained.


## 3. PPARγ: Mediator of Development and Disease in Bone Biology

As discussed previously, maintenance
of bone homeostasis is mediated through a balance of osteoblast-mediated bone
deposition and osteoclast-mediated bone resorption. The continued production of
these cells from stromal (mesenchymal) and hematopoietic (monocyte) precursors,
respectively, is an essential component in the maintenance of BMD. Stromal
progenitor or mesencymal stem cells are multipotent cells, capable of producing
cells of a number of different lineages, including osteoblasts and adipocytes [47–49].Since the early 1990s, researchers have
hypothesized that a “see-saw” relationship exists in the bone marrow cavity,
where production of adipocytes from stromal precursors is at the expense of osteoblast
production and vice versa [50, 51]. This theory is born out by a clinically observed
phenomenon, such as the increased adipocyte content of osteoporotic and aging
bone [51–53] as well as
studies where agents inducing adipocyte production reduced osteoblast number
[49, 50]. Likewise, treatment of bone marrow stromal cells with bone
morphogenic proteins (BMPs)
resulted in reduced formation of adipocytes [53]. Adipocytes can also produce
secreted factors such leptin and estrogen, which can positively regulate bone
mass [13, 54, 55], further underlining the interrelated nature of bone and fat
development (see Figure 1).PPARs are ligand-activated
nuclear hormone receptors which stimulate expression of genes containing peroxisome
proliferator response elements (PPREs)  [53, 54]. There
are three principal members of this family, PPARα, PPARδ,
and PPARγ,
activation of which stimulates genes involved in fatty acid oxidation,
uncoupling of respiration toward heat production (thermoregulation) and
terminal adipocyte differentiation (including intracellular lipid accumulation),
respectively, (see Table 1)  [49, 50, 55–59].Table 1
PPARγ-regulated genes involved in
adiogenesis, glucose uptake, and thermoregulation (↑ positive regulation; ↓
negative regulation).
PPARγ-regulated genesGeneTissue/cell typeFunctionCCAT enhancer binding proteinα (CEBPα) [7]↑Adipose/preadipose tissueTranscription factor. CDK2/4 inhibition-cell cycle arrestAdipose differentiation related protein (ADRP) [50]↑Adipose/preadipose tissueAssociated with globule membrane, early marker of adipocyte differentiationLipoprotein lipase [49]↑Vascular endothelium, heart, muscle, adiposeLipid hydrolysis from lipoproteinsAdiponectin [49]↑Adipose tissue (secreted)Fatty acid catabolismAdipocyte protein 2 (aP2/FABP4)
[49]↑Adipocytes/macrophagesIntercellular lipid transportTumour suppressor candidate 5 (TUSC 5) [56]↑Preadipose/adipose tissueAssociated with entry into the
later stages of adipogenesisGlucose transporters 4 (GLUT) 4  [57]↑Wide tissue distributionInsulin stimulated glucose uptakeUncoupling proteins 1-3 (UCP 1-3)  [58]↑Adipose tissue, skeletal muscle, liverThermogenesis/thermoregulationThe activity of PPARγ and RUNX-2 is
a key to our understanding of the relationship between fat and bone. Activity
of the RUNX-2 transcription factor is not only essential for maintenance of osteoblast
phenotype, but it is
also involved in driving the differentiation of osteoblasts from mesenchymal
stem cells [11–14], while activity
of PPARγ in mesenchymal stem cells induces
differentiation into adipocytes. The eventual phenotype of the differentiating
cell is generally considered to be controlled by an antagonistic balance
between RUNX-2 and PPARγ
[13, 14]. Studies have
demonstrated, for example, that activation of PPARγ using pharmacological agents can lead to decreased bone mass in vivo, while mice lacking the PPARγ gene display increased bone mass and an inability to develop adipocytes
[59–61]. Indeed, even in the eventual mature
cell, the function can be altered by dysregulating this balance, with in
vivo studies using a mouse
model demonstrating reduced bone formation rate and suppression of RUNX-2 in osteoblasts
in which PPARγ had been activated [61], while Kim et al. have demonstrated that activation
of PPARγ induces death through a MAPK-dependant
mechanism in osteoblastic cells [62].PPARγ deficient mice (having
a mutation in the PPARγ2 locus) have been generated and display a “lipodystrophic” phenotype, which occurs concurrently
with increased bone mass, to the point where the bone marrow is almost
completely occluded and hematopoiesis moves to extramedullary sites, such as the spleen
[61, 63]. Recently, our understanding of the roles of PPARγ in numerous physiologic processes, including the bone/fat paradigm, has
been furthered by the development of the thiazolidinedione (TDZ) family of PPARγ ligands, such as netoglitazone, pioglitazone, rosiglitazone, and GW0072
[64–67]. Studies have demonstrated that treatment of murine osteoblasts with
netoglitazone and GW0072 can block osteoblast differentiation, without inducing
adipogenesis [62, 64], while in vivo
studies have demonstrated that rosiglitazone, a ligand with higher affinity for
PPARγ, decreased bone mineral density,
bone formation rate, and trabecular bone volume, while increasing adipogenesis
[65, 67]. Further studies on ovariectomized rats revealed that these effects
are mediated in part by the suppression of the RUNX-2 transcription factor [67],
giving further strength to the argument that an antagonistic relationship
between PPARγ and RUNX-2 governs bone and fat formation. Indeed,
Hong et al. have demonstrated that shared coactivator protein, TAZ, accounts
in some part for this relationship, in that it coactivates RUNX-2 and bone
formation, while suppressing PPARγ
[68].


### 3.1. PPARγ in HIV-1-Associated Lipodystrophy

ART is associated with changes in
fat metabolism, broadly termed lipodystrophy (changes in fat distribution) or
lipoatrophy (atrophy of adipose tissue). Severe forms of lipodystrophy are a
major cosmetic concern, and can lead to suboptimal adherence to therapy. In
addition, lipodystrophy is associated with markers of cardiovascular risk, such
as insulin resistance and dyslipidemia [5].In vitro, expression of PPARγ is decreased by exposure to anti-HIV-1 PI and
NRTI drugs. In differentiating
adipocytes, exposure to nelfinavir, saquinavir, and ritonavir at 10 μM
concentrations resulted in decreased adipogenesis and expression of the PPARγ-mediated mRNA encoding aP2 and lipoprotein lipase (LPL)  [10]. Similar effects on PPARγ expression were observed in 3T3-F442A
adipocyte cells exposed to 10–50 μM indinavir [69], while studies by the same group have also
demonstrated that the nuclear association of the PPARγ regulator SREBP-1 is
reduced by treatment with indinavir [70]. In
mature adipocytes, inhibition of PPARγ function by expression of a dominant
negative PPARγ isoform results in decreased accumulation of intracellular
triglyceride, decreased cell size, and decreased expression of genes involved in
both fatty acid
and glucose metabolism, including the glucose transporter GLUT-4 [71]. In
lipoatrophic mice, ablation of PPARγ activity
in liver resulted in hepatic steatosis, hypertriglyceridemia, and muscle insulin
resistance [72]. Many of these features are shared by PI-treated patients with HIV-1-associated
lipodystrophy.In vivo, patients with lipodystrophy
had lower adipose tissue expression of both PPARδ and PPARγ than those without lipodystrophy. This was accompanied
by decreases in a number of PPARγ-responsive
downstream genes including LPL and GLUT-4 [73, 74]. In studies by Mallon, NRTI treatment of non-HIV-1-infected subjects (either stavudine/lamivudine or
zidovudine/lamivudine
for six weeks) resulted in reduced PPARγ expression in adipose tissue (alongside alterations in transcription of
mitochondrial DNA, and upregulation of genes associated with mitochondrial
transcriptional regulation), although in this study the effects on overall fat
mass were not determined [9].In patients with
type 2 diabetes, exposure to TZD, which act as PPARγ ligands, resulted in increased expression of
PPARγ-target
genes such as LPL and fatty acid synthase (FAS) in subcutaneous adipose tissue
biopsies, without increasing expression of PPARγ itself [71]. However, studies utilizing TZD to
treat lipodystrophy have produced variable, and at best, modest results [75–78]. More
recently, van Wijk et al.
demonstrated that rosiglitazone treatment, compared to treatment with metformin, increased
subcutaneous abdominal and visceral abdominal fat in lipodystrophy, however
this was a small study (n=39), was not blinded or placebo controlled, and did not
measure clinical outcomes [79].The weight
clinical and scientific evidence suggests that HIV-1/ART-associated lipid abnormalities occur largely as a
result of treatment rather than infection. However, a recent study raised the
possibility that there may also be a viral component; Shrivastav et al. [80] demonstrated that treatment
with the HIV-1 accessory viral protein R (Vpr) could suppress PPARγ-induced transactivation
in 3T3-L1 murine adipocyte cells, with a consequent inhibition of adipocyte
differentiation. Vpr is a 96-amino-acid
accessory protein, which is packaged in the viral capsid, and is found in the
nucleus early after cell infection [81, 82]. Among the functions of Vpr is its
ability to act as a transcriptional activator of viral and cellular promoters [83–86]. Vpr enhances
the activity of steroid hormone receptors, including the gluticorticoid
receptor (GR), which Vpr can bind via its LXXLL motif [85]. Studies involving cotransfection with
constructs expressing wild type and mutant (LXXLL null) Vpr constructs with
reporter constructs containing the PPRE demonstrated that this phenomenon was dependent
on the LXXLL motif. Further experiments demonstrated that the GR did not play a
role, and that Vpr and PPARγ interacted directly in living cells.
The authors of this study hypothesize that in vivo circulating Vpr, or Vpr produced as a
result of direct infection of adipocytes, could suppress differentiation of
preadipocytes in a PPARγ dependent manner with obvious
consequences for the development of lipodystrophy and insulin resistance [80].


### 3.2. PPARγ in HIV-1-Associated Bone Disease

In contrast to the clearly defined role for PPARγ in
HIV-1/ART-associated lipid abnormalities, few studies have focused on its
potential impact in HIV-1/ART-associated bone abnormalities.To date, studies into mechanism of reduced bone
density have been understandably focused on two distinct strands, namely, the
effects on osteoblast and osteoclast number and function. In the case of OC
research, several studies have demonstrated that osteoclast function can be
altered in vitro by treatment
with both ritonavir and HIV-1 gp120 [87, 88]. Jain et al. demonstrated
that osteoclast activity, measured using a rat neonatal calvaria assay,
increased in the presence of nelfinavir, indinavir, saquinavir, or ritonavir, while
lopinavir and amprenavir did not increase osteoclast activity. In addition, Pan
et al. reported a significant
increase in markers of osteoclastogenesis (namely, the activity of the tartaric
acid phosphatase (TRAP) promoter and the NF-κb
transcription factor) in RAW264.7 (mouse leukemic
monocyte macrophage cell line
cells) and primary mouse osteoclast precursors treated with the NRTI zidovudine [89]. This same
group has more recently reported that the NRTIs ddi and lamiduvine also induced
osteoclastogenesis in vitro and
osteopenia in an in vivo mouse
model [90].Similarly, osteoblast-based studies have produced
some interesting data. Clinically,  Serrano et al. reported
reduced numbers of osteoclasts in HIV patients; a phenomenon occurring along
side-reduced serum osteocalcin levels and bone formation rate [19]. Previous
and ongoing in vitro studies by our own group have
demonstrated that osteoblast activity (as measured by calcium deposition and
alkaline phosphatase activity) can be reduced by a number of antiretroviral
drugs (including both
nelfinavir and indinavir). In addition, these studies identified tissue
inhibitor of metalloproteinase-3 (TIMP-3) as a mechanism for this observed loss
in osteoblast function [91]. Further studies by our group demonstrated that
treatment with the HIV-1 proteins p55-gag and gp120 reduced osteoblast activity
in conjunction with reduction RUNX-2 transcription factor activity [92]. Interestingly,
gp120 both decreased RUNX-2 activity
and increased PPARγ. Furthermore, our studies
investigating the effect of HIV-1 proteins on mesenchymal stem cell
differentiation have suggested that the proteins p55 and REV alter both mesenchymal
stem cell osteoblastic differentiation and RUNX-2/PPARγ signalling in nondifferentiating mesenchymal stem cells [93].Although these studies used a somewhat simplistic model of HIV-1 exposure, given the
evidence of the impact of PPARγ on normal bone biology, and the observation
that it can be perturbed in HIV-1-associated lipodystrophy, it is tempting to
interpret these results as being suggestive of PPARγ playing a role in HIV-1-mediated
bone disease. However, there is an obvious stumbling block for this hypothesis,
namely, that if increased PPARγ activity in mesenchymal stem cell
and osteoblasts could result in reduced bone mass, it would surely also
increase fat mass.
This picture is further complicated, as previously discussed studies
have demonstrated that treatment of non-HIV-1-infected subjects with NNRTIs
resulted in reduced PPARγ expression in adipose tissue [9],
while in vitro studies with
3T3-F442A cells have demonstrated that both PPARγ expression and its association with SREBP-1 are reduced by treatment
with indinavir [69, 70]. However, different processes may govern fat
redistribution in different tissues, with gain in visceral fat and loss of
subcutaneous fat. In addition, at least one ex vivo study suggests that both markers of adipocyte and osteoblastic
differentiation are significantly reduced in human mesenchymal stem cells
treated with a subset of protease inhibitors (particularly nelfinavir and saquinavir)  [94], while HIV-1 patients receiving the NRTI zidovudine were shown to have
reduced both BMD and whole body fat [33]. Could it be that contributing to both
HIV-1/ART-associated bone and lipid disorders is an underlying disregulation of
mesenchymal stem cell function combined with separate effects on adult or
partially differentiated cells?


## 3.1. PPARγ in HIV-1-Associated Lipodystrophy

ART is associated with changes in
fat metabolism, broadly termed lipodystrophy (changes in fat distribution) or
lipoatrophy (atrophy of adipose tissue). Severe forms of lipodystrophy are a
major cosmetic concern, and can lead to suboptimal adherence to therapy. In
addition, lipodystrophy is associated with markers of cardiovascular risk, such
as insulin resistance and dyslipidemia [5].In vitro, expression of PPARγ is decreased by exposure to anti-HIV-1 PI and
NRTI drugs. In differentiating
adipocytes, exposure to nelfinavir, saquinavir, and ritonavir at 10 μM
concentrations resulted in decreased adipogenesis and expression of the PPARγ-mediated mRNA encoding aP2 and lipoprotein lipase (LPL)  [10]. Similar effects on PPARγ expression were observed in 3T3-F442A
adipocyte cells exposed to 10–50 μM indinavir [69], while studies by the same group have also
demonstrated that the nuclear association of the PPARγ regulator SREBP-1 is
reduced by treatment with indinavir [70]. In
mature adipocytes, inhibition of PPARγ function by expression of a dominant
negative PPARγ isoform results in decreased accumulation of intracellular
triglyceride, decreased cell size, and decreased expression of genes involved in
both fatty acid
and glucose metabolism, including the glucose transporter GLUT-4 [71]. In
lipoatrophic mice, ablation of PPARγ activity
in liver resulted in hepatic steatosis, hypertriglyceridemia, and muscle insulin
resistance [72]. Many of these features are shared by PI-treated patients with HIV-1-associated
lipodystrophy.In vivo, patients with lipodystrophy
had lower adipose tissue expression of both PPARδ and PPARγ than those without lipodystrophy. This was accompanied
by decreases in a number of PPARγ-responsive
downstream genes including LPL and GLUT-4 [73, 74]. In studies by Mallon, NRTI treatment of non-HIV-1-infected subjects (either stavudine/lamivudine or
zidovudine/lamivudine
for six weeks) resulted in reduced PPARγ expression in adipose tissue (alongside alterations in transcription of
mitochondrial DNA, and upregulation of genes associated with mitochondrial
transcriptional regulation), although in this study the effects on overall fat
mass were not determined [9].In patients with
type 2 diabetes, exposure to TZD, which act as PPARγ ligands, resulted in increased expression of
PPARγ-target
genes such as LPL and fatty acid synthase (FAS) in subcutaneous adipose tissue
biopsies, without increasing expression of PPARγ itself [71]. However, studies utilizing TZD to
treat lipodystrophy have produced variable, and at best, modest results [75–78]. More
recently, van Wijk et al.
demonstrated that rosiglitazone treatment, compared to treatment with metformin, increased
subcutaneous abdominal and visceral abdominal fat in lipodystrophy, however
this was a small study (n=39), was not blinded or placebo controlled, and did not
measure clinical outcomes [79].The weight
clinical and scientific evidence suggests that HIV-1/ART-associated lipid abnormalities occur largely as a
result of treatment rather than infection. However, a recent study raised the
possibility that there may also be a viral component; Shrivastav et al. [80] demonstrated that treatment
with the HIV-1 accessory viral protein R (Vpr) could suppress PPARγ-induced transactivation
in 3T3-L1 murine adipocyte cells, with a consequent inhibition of adipocyte
differentiation. Vpr is a 96-amino-acid
accessory protein, which is packaged in the viral capsid, and is found in the
nucleus early after cell infection [81, 82]. Among the functions of Vpr is its
ability to act as a transcriptional activator of viral and cellular promoters [83–86]. Vpr enhances
the activity of steroid hormone receptors, including the gluticorticoid
receptor (GR), which Vpr can bind via its LXXLL motif [85]. Studies involving cotransfection with
constructs expressing wild type and mutant (LXXLL null) Vpr constructs with
reporter constructs containing the PPRE demonstrated that this phenomenon was dependent
on the LXXLL motif. Further experiments demonstrated that the GR did not play a
role, and that Vpr and PPARγ interacted directly in living cells.
The authors of this study hypothesize that in vivo circulating Vpr, or Vpr produced as a
result of direct infection of adipocytes, could suppress differentiation of
preadipocytes in a PPARγ dependent manner with obvious
consequences for the development of lipodystrophy and insulin resistance [80].


## 3.2. PPARγ in HIV-1-Associated Bone Disease

In contrast to the clearly defined role for PPARγ in
HIV-1/ART-associated lipid abnormalities, few studies have focused on its
potential impact in HIV-1/ART-associated bone abnormalities.To date, studies into mechanism of reduced bone
density have been understandably focused on two distinct strands, namely, the
effects on osteoblast and osteoclast number and function. In the case of OC
research, several studies have demonstrated that osteoclast function can be
altered in vitro by treatment
with both ritonavir and HIV-1 gp120 [87, 88]. Jain et al. demonstrated
that osteoclast activity, measured using a rat neonatal calvaria assay,
increased in the presence of nelfinavir, indinavir, saquinavir, or ritonavir, while
lopinavir and amprenavir did not increase osteoclast activity. In addition, Pan
et al. reported a significant
increase in markers of osteoclastogenesis (namely, the activity of the tartaric
acid phosphatase (TRAP) promoter and the NF-κb
transcription factor) in RAW264.7 (mouse leukemic
monocyte macrophage cell line
cells) and primary mouse osteoclast precursors treated with the NRTI zidovudine [89]. This same
group has more recently reported that the NRTIs ddi and lamiduvine also induced
osteoclastogenesis in vitro and
osteopenia in an in vivo mouse
model [90].Similarly, osteoblast-based studies have produced
some interesting data. Clinically,  Serrano et al. reported
reduced numbers of osteoclasts in HIV patients; a phenomenon occurring along
side-reduced serum osteocalcin levels and bone formation rate [19]. Previous
and ongoing in vitro studies by our own group have
demonstrated that osteoblast activity (as measured by calcium deposition and
alkaline phosphatase activity) can be reduced by a number of antiretroviral
drugs (including both
nelfinavir and indinavir). In addition, these studies identified tissue
inhibitor of metalloproteinase-3 (TIMP-3) as a mechanism for this observed loss
in osteoblast function [91]. Further studies by our group demonstrated that
treatment with the HIV-1 proteins p55-gag and gp120 reduced osteoblast activity
in conjunction with reduction RUNX-2 transcription factor activity [92]. Interestingly,
gp120 both decreased RUNX-2 activity
and increased PPARγ. Furthermore, our studies
investigating the effect of HIV-1 proteins on mesenchymal stem cell
differentiation have suggested that the proteins p55 and REV alter both mesenchymal
stem cell osteoblastic differentiation and RUNX-2/PPARγ signalling in nondifferentiating mesenchymal stem cells [93].Although these studies used a somewhat simplistic model of HIV-1 exposure, given the
evidence of the impact of PPARγ on normal bone biology, and the observation
that it can be perturbed in HIV-1-associated lipodystrophy, it is tempting to
interpret these results as being suggestive of PPARγ playing a role in HIV-1-mediated
bone disease. However, there is an obvious stumbling block for this hypothesis,
namely, that if increased PPARγ activity in mesenchymal stem cell
and osteoblasts could result in reduced bone mass, it would surely also
increase fat mass.
This picture is further complicated, as previously discussed studies
have demonstrated that treatment of non-HIV-1-infected subjects with NNRTIs
resulted in reduced PPARγ expression in adipose tissue [9],
while in vitro studies with
3T3-F442A cells have demonstrated that both PPARγ expression and its association with SREBP-1 are reduced by treatment
with indinavir [69, 70]. However, different processes may govern fat
redistribution in different tissues, with gain in visceral fat and loss of
subcutaneous fat. In addition, at least one ex vivo study suggests that both markers of adipocyte and osteoblastic
differentiation are significantly reduced in human mesenchymal stem cells
treated with a subset of protease inhibitors (particularly nelfinavir and saquinavir)  [94], while HIV-1 patients receiving the NRTI zidovudine were shown to have
reduced both BMD and whole body fat [33]. Could it be that contributing to both
HIV-1/ART-associated bone and lipid disorders is an underlying disregulation of
mesenchymal stem cell function combined with separate effects on adult or
partially differentiated cells?


## 4. Conclusion

The importance of PPARγ in both bone and fat metabolism has
been clearly demonstrated, and while a role for PPARγ in the lipid abnormalities associated with HIV-1 and its treatment is
emerging, its involvement in HIV-1-associated bone disease remains unclear.
Given the common origin of both adipocytes and osteoblasts from mesenchymal
stem cell, and the demonstrated effect of increased PPARγ expression on bone in vitro
and in vivo, we hypothesize a
potential role for PPARγ in the reduced bone mass associated
with HIV-1 infection and treatment. It may be possible that HIV-1 infection
and/or treatment, through dysregulating PPARγ (and possibly also RUNX-2) activity in undifferentiated stromal cells,
or in partially differentiated preosteoblast and preadipocyte cells, can reduce
the eventual number or functional capacity of the adult cell types.In order to further investigate this hypothesis, it may be worthwhile to conduct ex vivo experiment on primary mesenchymal
stem cells collected from HIV-1 patients. The expression and activity of PPARγ and differentiation potential of these cells could be assessed and
compared to those of cells harvested from uninfected individuals, and the data
gathered used to generate a new model of HIV-1/PPARγ/mesenchymal stem cell interactions.It is clear that further studies are necessary to more fully describe the role
of PPARγ in the setting of HIV-1-associated
bone disease and its interplay with vascular and fat disorders.



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