Anemia in Patients With Resistance to Thyroid Hormone α: A Role for Thyroid Hormone Receptor α in Human Erythropoiesis

Context: Patients with resistance to thyroid hormone (TH) α (RTHα) are characterized by growth retardation, macrocephaly, constipation, and abnormal thyroid function tests. In addition, almost all RTHα patients have mild anemia, the pathogenesis of which is unknown. Animal studies suggest an important role for TH and TH receptor (TR)α in erythropoiesis.Objective: To investigate whether a defect in TRα affects the maturation of red blood cells in RTHα patients.Design, Setting, and Patients: Cultures of primary human erythroid progenitor cells (HEPs), from peripheral blood of RTHα patients (n = 11) harboring different inactivating mutations in TRα (P398R, F397fs406X, C392X, R384H, A382fs388X, A263V, A263S), were compared with healthy controls (n = 11). During differentiation, erythroid cells become smaller, accumulate hemoglobin, and express different cell surface markers. We assessed cell number and cell size, and used cell staining and fluorescence-activated cell sorter analysis to monitor maturation at different time points.Results: After ∼14 days of ex vivo expansion, both control and patient-derived progenitors differentiated spontaneously. However, RTHα-derived cells differentiated more slowly. During spontaneous differentiation, RTHα-derived HEPs were larger, more positive for c-Kit (a proliferation marker), and less positive for glycophorin A (a differentiation marker). The degree of abnormal spontaneous maturation of RTHα-derived progenitors did not correlate with severity of underlying TRα defect. Both control and RTHα-derived progenitors responded similarly when differentiation was induced. T3 exposure accelerated differentiation of both control- and RTHα patient-derived HEPs.Conclusions: Inactivating mutations in human TRα affect the balance between proliferation and differentiation of progenitor cells d

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

TSH elevations as the first laboratory evidence for pseudohypoparathyroidism type Ib (PHP-Ib).

Hypocalcemia and hyperphosphatemia because of resistance toward parathyroid hormone (PTH) in the proximal renal tubules are the most prominent abnormalities in patients affected by pseudohypoparathyroidism type Ib (PHP-Ib). In this rare disorder, which is caused by GNAS methylation changes, resistance can occur toward other hormones, such as thyroid-stimulating hormone (TSH), that mediate their actions through G protein-coupled receptors. However, these additional laboratory abnormalities are usually not recognized until PTH-resistant hypocalcemia becomes clinically apparent. We now describe four pediatric patients, first diagnosed with subclinical or overt hypothyroidism between the ages of 0.2 and 15 years, who developed overt PTH-resistance 3 to 20 years later. Although anti-thyroperoxidase (anti-TPO) antibodies provided a plausible explanation for hypothyroidism in one of these patients, this and two other patients revealed broad epigenetic GNAS abnormalities, which included loss of methylation (LOM) at exons AS, XL, and A/B, and gain of methylation at exon NESP55; ie, findings consistent with PHP-Ib. LOM at GNAS exon A/B alone led in the fourth patient to the identification of a maternally inherited 3-kb STX16 deletion, a well-established cause of autosomal dominant PHP-Ib. Although GNAS methylation changes were not detected in additional pediatric and adult patients with subclinical hypothyroidism (23 pediatric and 39 adult cases), hypothyroidism can obviously be the initial finding in PHP-Ib patients. One should therefore consider measuring PTH, along with calcium and phosphate, in patients with unexplained hypothyroidism for extended periods of time to avoid hypocalcemia and associated clinical complications

Anemia in Patients With Resistance to Thyroid Hormone α: A Role for Thyroid Hormone Receptor α in Human Erythropoiesis

Context: Patients with resistance to thyroid hormone (TH) α (RTHα) are characterized by growth retardation, macrocephaly, constipation, and abnormal thyroid function tests. In addition, almost all RTHα patients have mild anemia, the pathogenesis of which is unknown. Animal studies suggest an important role for TH and TH receptor (TR)α in erythropoiesis. Objective: To investigate whether a defect in TRα affects the maturation of red blood cells in RTHα patients. Design, Setting, and Patients: Cultures of primary human erythroid progenitor cells (HEPs), from peripheral blood of RTHα patients (n = 11) harboring different inactivating mutations in TRα (P398R, F397fs406X, C392X, R384H, A382fs388X, A263V, A263S), were compared with healthy controls (n = 11). During differentiation, erythroid cells become smaller, accumulate hemoglobin, and express different cell surface markers. We assessed cell number and cell size, and used cell staining and fluorescence-activated cell sorter analysis to monitor maturation at different time points. Results: After ∼14 days of ex vivo expansion, both control and patient-derived progenitors differentiated spontaneously. However, RTHα-derived cells differentiated more slowly. During spontaneous differentiation, RTHα-derived HEPs were larger, more positive for c-Kit (a proliferation marker), and less positive for glycophorin A (a differentiation marker). The degree of abnormal spontaneous maturation of RTHα-derived progenitors did not correlate with severity of underlying TRα defect. Both control and RTHα-derived progenitors responded similarly when differentiation was induced. T3 exposure accelerated differentiation of both control- and RTHα patient-derived HEPs. Conclusions: Inactivating mutations in human TRα affect the balance between proliferation and differentiation of progenitor cells during erythropoiesis, which may contribute to the mild anemia seen in most RTHα patients.A.L.M.v.G., M.E.M., and R.P.P. are supported by ZonMWTOP Grant 91212044 and an Erasmus MC Medical Research Advisory Committee (MRACE) grant. A.L.M.v.G. and R.P.P. are also supported by a European Thyroid Association (ETA) research grant. K. Chatterjee is supported by Wellcome Trust Investigator Award 095564/Z/11/Z. K. Chatterjee and C.M. are supported by the National Institute for Health Research Cambridge Biomedical Research Centre

The role of apoptosis in growth plate cartilage during normal and abnormal growth

Longitudinal bone growth occurs at the growth plate where resting chondrocytes proliferate, differentiate into a hypertrophic form, and finally become terminal hypertrophic chondrocytes before giving rise to bone. Because growth occurs when proliferation exceeds cell death, alterations in cell death by apoptosis, which is essential for the maintenance of tissue homeostasis, could affect growth. Therefore, apoptosis could be an important regulator of growth plate homeostasis and maturation, and subsequently linear growth. As an example, it has been proposed that the fate of the terminal hypertrophic chondrocytes is death by apoptosis but this is still debated. Furthermore, apoptosis of proliferative chondrocytes could be a mechanism of growth retardation in growth inhibiting conditions. We first studied apoptosis during normal growth plate maturation. Our studies revealed that apoptosis and apoptosis related proteins are developmentally regulated. When the growth plate matures and the growth rate decreases, apoptosis is increased and is observed mainly in terminal hypertrophic chondrocytes at all developmental stages. The developmental changes in apoptosis occurred concurrently with changes in the Bcl family of proteins and caspases, supporting the apoptosis data. The observed developmental changes in apoptosis of terminal hypertrophic chondrocytes and their limited number may suggest that the fate of those cells is not always death by classical apoptosis. To investigate whether apoptosis is a mechanism of growth retardation in growthinhibiting conditions, we chose glucocorticoid-induced growth retardation as a model. Growth plates from rats treated with dexamethasone showed increased apoptosis mainly in terminal hypertrophic chondrocytes but also in early proliferative cells. Apoptosis was associated with decreased immunoreactivity for the anti-apoptotic proteins Bcl-2 and BcI-X, and increased immunoreactivity for caspase-3, thus supporting the apoptosis data. Furthermore, because Bcl-2 lies downstream of the PTHrP signalling pathway, we immunolocalized PTHrP, which proved to be down-regulated by dexamethasone. Our data suggest that apoptosis is a mechanism of growth retardation in dexamethasone-induced apoptosis. The premature loss of proliferative chondrocytes by apoptosis could diminish the growth potential. We used the HCS-2/8 chondrocytic cell line to examine dexamethasone-induced apoptosis of proliferative chondrocytes, the underlying mechanisms and the possible anti-apoptotic role of IGF-I. Dexamethasone induced apoptosis in a dose dependent manner after 48 and 72 hours in culture through inhibition of Akt phosphorylation. Co-culture with IGF-I protected cells from dexamethasone-induced apoptosis, most likely through signalling pathways other than Akt/Pl3K. Apoptosis was caspase-dependent, with caspase-8 activation preceding that of caspase-9. We also studied the role of apoptosis in the growth plate in cytokine-induced growth retardation. In an organ culture of fetal rat metatarsals we found that IL- I and TNF alone severely inhibited metatarsal growth, with synergistic effects when combined. In addition to decreased proliferation, apoptosis was markedly increased in proliferative chondrocytes. Antibodies against IL-1 or TNF, and co-culture with IGF-I improved growth and decreased apoptosis. The marked loss of proliferative cells by apoptosis could explain the severe growth retardation and incomplete catch-up growth observed in chronic inflammatory conditions

Insulin-Like Growth Factor-I Overexpression Attenuates Cerebellar Apoptosis by Altering the Expression of Bcl Family Proteins in a Developmentally Specific Manner

In studies of transgenic (Tg) mice that overexpress insulin-like growth factor-I (IGF-I) exclusively in the CNS, we demonstrated a dramatic increase in cerebellar granule cell number that appeared to be attributable predominantly to enhanced survival. IGF-I anti-apoptotic actions are well established in cultured neurons, but comparable studies in vivo are few. Using the same Tg mice, therefore, we set out to document IGF-I antiapoptotic effects during cerebellar development and to probe IGF-I signaling mechanisms. Compared with cerebella (CBs) of non-Tg littermates, those of Tg mice had fewer apoptotic cells at postnatal day 7 (P7) and showed a similar tendency at P14 and P21. At each age studied, procaspase-3 and caspase-3 were decreased in CBs of Tg mice. The caspase-3 decline was accompanied by decreases in the 85 kDa fragment of Poly(ADP-ribose) polymerase, a known product of caspas

Myostatin serum levels in children with type 1 diabetes mellitus

Purpose Type 1 diabetes mellitus (T1DM) can cause several complications, among them myopathy, which can appear even in adolescents. This is of importance, since skeletal muscle is the largest of the insulin-sensitive tissues and thus plays a significant role in glucose homeostasis. A prime regulator of skeletal muscle mass is myostatin, a protein which has a negative role in skeletal muscle development but also in glucose homeostasis, causing insulin resistance. Since myopathy is a complication of T1DM and myostatin is a fundamental regulator of skeletal muscle and is also involved in glucose homeostasis, we investigated the serum levels of myostatin in children with T1DM. Methods We determined myostatin serum levels using ELISA in 87 children with T1DM aged 10.62 +/- 3.94 years, and in 75 healthy children aged 10.46 +/- 3.32 years old. Results My omicron statin was significantly elevated in T1DM compared to the healthy control children (23.60 +/- 7.70 vs 16.74 +/- 6.95 ng/ml, p < 0.0001). Myostatin was not correlated with body mass index (BMI) SD or hemoglobin A1c (HbA1c). Conclusion Children with T1DM have significantly higher serum levels of myostatin compared to healthy children of the same age and BMI SD. The elevated myostatin in T1DM could reflect impaired muscle function and/or glucose metabolism, or could represent a homeostatic mechanism

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