Farasat Zaman

Proinsulin C-peptide Regulates Ribosomal RNA Expression

Proinsulin C-peptide is internalized into cells, but a function of its intracellular localization has not been established. We now demonstrate that, upon cellular entry, C-peptide is localized to the nucleoli, where it promotes transcription of genes encoding for ribosomal RNA. We find that C-peptide binds to histones and enhances acetylation of lysine residue 16 of histone H4 at the promoter region of genes for ribosomal RNA. In agreement with synchrony of ribosomal RNA synthesis and cell proliferation, we show that C-peptide stimulates proliferation in chondrocytes and HEK-293 cells. This regulation of ribosomal RNA provides a mechanism by which C-peptide can exert transcriptional effects and implies that the peptide has growth factor activity.

RECENT RESEARCH ON THE GROWTH PLATE: Impact of inflammatory cytokines on longitudinal bone growth

Children with inflammatory diseases usually display abnormal growth patterns as well as delayed puberty. This is a result of several factors related to the disease itself, such as malnutrition, hypercortisolism, and elevated levels of pro-inflammatory cytokines. These factors in combination with glucocorticoid treatment contribute to growth retardation during chronic inflammation by systemically affecting the major regulator of growth, the GH/IGF1 axis. However, recent studies have also shown evidence of a direct effect of these factors at the growth plate level. In conditions of chronic inflammation, pro-inflammatory cytokines are upregulated and released into the circulation. The most abundant of these, tumor necrosis factor α, interleukin 1β (IL1β), and IL6, are all known to directly act on growth plate cartilage to induce apoptosis and thereby suppress bone growth. Both clinical and experimental studies have shown that growth retardation can partly be rescued when these cytokines are blocked. Therefore, therapy modulating the local actions of these cytokines may be effective for preventing growth failure in patients with chronic inflammatory disorders. In this review, we report the current knowledge of inflammatory cytokines and their role in regulating bone growth.

Proteasome Inhibition Up-regulates p53 and Apoptosis-Inducing Factor in Chondrocytes Causing Severe Growth Retardation in Mice

Proteasome inhibitors (PI), a novel class of anticancer drugs, are relatively well tolerated and have recently been introduced into the clinic for the treatment of multiple myeloma. The tumor selectivity and low toxicity of PIs are surprising, given the crucial role of the ubiquitin/proteasome system in a multitude of cellular processes. Here, we show that systemic administration of PIs specifically impairs the ubiquitin/proteasome system in growth plate chondrocytes. Importantly, young mice displayed severe growth retardation during treatment as well as 45 days after the cessation of treatment with clinically relevant amounts of MG262 (0.2 micromol/kg body weight/injection) or bortezomib (1.0 mg/kg body weight/injection). Dysfunction of the ubiquitin/proteasome system was accompanied by the induction of apoptosis of stem-like and proliferative chondrocytes in the growth plate. These results were recapitulated in cultured fetal rat metatarsal bones and chondrocytic cell lines (rat, human). Apoptosis was associated with up-regulation of the proapoptotic molecules, p53 and apoptosis-inducing factor (AIF), both in vitro and in vivo. In addition to the observation that AIF is expressed in the growth plate, we also provide evidence that AIF serves as a direct target protein for ubiquitin, thus explaining its prominent up-regulation upon proteasome inhibition. Suppression of p53 or AIF expression with small interfering RNAs partly rescued chondrocytes from proteasome inhibition-induced apoptosis (35% and 41%, respectively). Our observations show that proteasome inhibition may selectively target essential cell populations in the growth plate causing significant growth failure. These findings could have important implications for the use of proteasome inhibitors in the treatment of childhood cancer.

Ablation of the pro-apoptotic protein Bax protects mice from glucocorticoid-induced bone growth impairment.

Dexamethasone (Dexa) is a widely used glucocorticoid to treat inflammatory diseases; however, a multitude of undesired effects have been reported to arise from this treatment including osteoporosis, obesity, and in children decreased longitudinal bone growth. We and others have previously shown that glucocorticoids induce apoptosis in growth plate chondrocytes. Here, we hypothesized that Bax, a pro-apoptotic member of the Bcl-2 family, plays a key role in Dexa-induced chondrocyte apoptosis and bone growth impairment. Indeed, experiments in the human HCS-2/8 chondrocytic cell line demonstrated that silencing of Bax expression using small-interfering (si) RNA efficiently blocked Dexa-induced apoptosis. Furthermore, ablation of Bax in female mice protected against Dexa-induced bone growth impairment. Finally, Bax activation by Dexa was confirmed in human growth plate cartilage specimens cultured ex vivo. Our findings could therefore open the door for new therapeutic approaches to prevent glucocorticoid-induced bone growth impairment through specific targeting of Bax.

Dexamethasone differentially regulates Bcl-2 family proteins in human proliferative chondrocytes: Role of pro-apoptotic Bid

Glucocorticoids (GCs) are widely used to treat inflammatory diseases and cancers. A multitude of undesired side effects have been reported in GC-treated patients including decreased linear bone growth. We have previously reported that GCs activate the caspase cascade and trigger Bax-mediated mitochondrial apoptosis in growth plate chondrocytes causing growth retardation in young mice. To further explore the role of mitochondrial apoptosis in GC-induced bone growth retardation, a number of pro- and anti-apoptotic proteins were studied in ex vivo cultures of human growth plate cartilage and human HCS-2/8 proliferative chondrocytes exposed to dexamethasone. Dexamethasone was found to increase the pro-apoptotic proteins Bcl-xS, Bad, and Bak as well as the proteolysis of Bid. Anti-Bid small interfering RNA partially rescued the chondrocytes from dexamethasone-induced apoptosis. Taken together, our data suggest that GC treatment differentially regulates Bcl-2 family member proteins to facilitate mitochondrial apoptosis in proliferative chondrocytes thereby contributing to GC-induced bone growth impairment. Prevention of this imbalance between pro- and anti-apoptotic Bcl-2 family proteins may provide a new strategy to protect from adverse effects of GCs on bone growth.

Bortezomib Is Cytotoxic to the Human Growth Plate and Permanently Impairs Bone Growth in Young Mice

Bortezomib, a novel proteasome inhibitor approved for the treatment of cancer in adults, has recently been introduced in pediatric clinical trials. Any tissue-specific side effects on bone development have to our knowledge not yet been explored. To address this, we experimentally studied the effects of bortezomib in vivo in young mice and in vitro in organ cultures of rat metatarsal bones and human growth plate cartilage, as well as in a rat chondrocytic cell line. We found that bortezomib while efficiently blocking the ubiquitin/proteasome system (UPS) caused significant growth impairment in mice, by increasing resting/stem-like chondrocyte apoptosis. Our data support a local action of bortezomib, directly targeting growth plate chondrocytes leading to decreased bone growth since no suppression of serum levels of insulin-like growth factor-I (IGF-I) was observed. A local effect of bortezomib was confirmed in cultured rat metatarsal bones where bortezomib efficiently caused growth retardation in a dose dependent and irreversible manner, an effect linked to increased chondrocyte apoptosis, mainly of resting/stem-like chondrocytes. The cytotoxicity of bortezomib was also evaluated in a unique model of cultured human growth plate cartilage, which was found to be highly sensitive to bortezomib. Mechanistic studies of apoptotic pathways indicated that bortezomib induced activation of p53 and Bax, as well as cleavage of caspases and poly-ADP-ribose polymerase (PARP) in exposed chondrocytes. Our observations, confirmed in vivo and in vitro, suggest that bone growth could potentially be suppressed in children treated with bortezomib. We therefore propose that longitudinal bone growth should be closely monitored in ongoing clinical pediatric trials of this promising anti-cancer drug.

Proteasome inhibition therapies in childhood cancer

We enjoyed the excellent and timely review by Terpos et al.1 in a recent issue of Leukemia on the effects of novel anti-myeloma agents on bone metabolism. In this review, the authors elaborate on the ubiquitin/proteasome system in bone, and the use of proteasome inhibitors (PIs) in myeloma bone disease. The authors emphasize that PIs, including bortezomib, act locally in the bone to stimulate osteoblast differentiation while inhibiting osteoclast formation and making them attractive agents for further investigation, as they may combine anti-myeloma activity with beneficial effects on bone. Indeed, recent in vitro and in vivo observations support the hypothesis that a direct stimulatory effect on the bone formation process could occur during bortezomib treatment.2 Terpos et al. propose prospective studies with specific clinical end points, such as bone mineral density, to be undertaken. However, when designing such studies, we believe that it is important to pay particular attention to the recent observation that systemic administration of PIs specifically impairs the ubiquitin/proteasome system in chondrocytes and growth plate cartilage.3

Interleukin-6 acts locally on the growth plate to impair bone growth

A recent clinical study in mothers with rheumatoid arthritis (RA) published in this journal reported that high maternal serum levels of interleukin-6 (IL-6) are associated with low birthweight deliveries.1 Low birth weight in turn is associated with impaired growth during childhood.2 Interestingly, overexpression of IL-6 leads to reduced bone growth in mice.3 Although the systemic effects of IL-6 are well understood, any local effects on the growth plate have not yet been clarified. Aiming to study the local actions of IL-6, we isolated fetal rat metatarsal bones and cultured them ex vivo for 12 days while monitoring bone growth. When exposed to IL-6 and its soluble receptor IL-6Rα (10+100 ng/mL), growth was decreased by 21.1% compared with control (p<0.001) (figure 1A). IL-6 mainly targeted the hypertrophic growth plate zone, reducing its height by 53.7% and area by 72.6% (p<0.05 vs control; figure 1B). These data further …

Animal models to explore the effects of glucocorticoids on skeletal growth and structure

Glucocorticoids (GCs) are effective for the treatment of many chronic conditions, but their use is associated with frequent and wide-ranging adverse effects including osteoporosis and growth retardation. The mechanisms that underlie the undesirable effects of GCs on skeletal development are unclear, and there is no proven effective treatment to combat them. An in vivo model that investigates the development and progression of GC-induced changes in bone is, therefore, important and a well-characterized pre-clinical model is vital for the evaluation of new interventions. Currently, there is no established animal model to investigate GC effects on skeletal development and there are pros and cons to consider with the different protocols used to induce osteoporosis and growth retardation. This review will summarize the literature and highlight the models and techniques employed in experimental studies to date.

Erratum: Proinsulin C-peptide regulates ribosomal RNA expression (The Journal of Biological Chemistry (2010) 285 (3462-3469) DOI: 10.1074/jbc.M109.053587)

DOI 10.1074/jbc.A109.053587 Proinsulin C-peptide regulates ribosomal RNA expression. Emma Lindahl, Ulrika Nyman, Farasat Zaman, Carina Palmberg, Anna Cascante, Jawed Shafqat, Masaharu Takigawa, Lars Sävendahl, Hans Jörnvall, and Bertrand Joseph PAGES 3467 AND 3468: During the preparation of Figs. 5B and 6A, irrelevant lanes were removed without indicating the borders between different sections of the same immunoblot or gels. These errors have now been corrected and do not affect the results or conclusions of this work. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 10, p. 4382, March 10, 2017