Heba M. Ismail

Phosphorylation of Histone H3 Thr-45 Is Linked to Apoptosis

Numerous post-translational modifications have been identified in histones. Most of these occur within the histone tails, but a few have been identified within the histone core sequences. Histone core post-translational modifications have the potential to directly modulate nucleosome structure and consequently DNA accessibility. Here, we identify threonine 45 of histone H3 (H3T45) as a site of phosphorylation in vivo. We find that phosphorylation of H3T45 (H3T45ph) increases dramatically in apoptotic cells, around the time of DNA nicking. To further explore this connection, we analyzed human neutrophil cells because they are short-lived cells that undergo apoptosis in vivo. Freshly isolated neutrophils contain very little H3T45ph, whereas cells cultured for 20 h possess significant amounts; the kinetics of H3T45ph induction closely parallel those of caspase-3 activation. Cytokine inhibition of neutrophil apoptosis leads to reduced levels of H3T45ph. We identify protein kinase C-δ as the kinase responsible for H3T45ph in vitro and in vivo. Given the nucleosomal position of H3T45, we postulate that H3T45ph induces structural change within the nucleosome to facilitate DNA nicking and/or fragmentation.

Identification of autoantibody-negative autoimmune type 2 diabetic patients.

OBJECTIVE Islet autoimmunity has long been recognized in the pathogenesis of type 1 diabetes and is becoming increasingly acknowledged as a component in the pathogenesis of type 2 diabetes. Islet reactive T cells and autoantibodies have been demonstrated in type 1 diabetes, whereas islet autoimmunity in type 2 diabetes has been limited to islet autoantibodies. In this study, we investigated whether islet reactive T cells might also be present in type 2 diabetic patients and how islet reactive T cells correlate with β-cell function. RESEARCH DESIGN AND METHODS Adult phenotypic type 2 diabetic patients (n = 36) were screened for islet reactive T-cell responses using cellular immunoblotting and five islet autoantibodies (islet cell antibody, GADA, insulin autoantibody, insulinoma-associated protein-2 autoantibody, and zinc transporter autoantibody). RESULTS We identified four subgroups of adult phenotypic type 2 diabetic patients based on their immunological status (Ab−T−, Ab+T−, Ab−T+, and Ab+T+). The Ab−T+ type 2 diabetic patients demonstrated T-cell responses similar to those of the Ab+T+ type 2 diabetic patients. Data were adjusted for BMI, insulin resistance, and duration of diabetes. Significant differences (P < 0.02) were observed among groups for fasting and glucagon-stimulated C-peptide responses. T-cell responses to islet proteins were also demonstrated to fluctuate less than autoantibody responses. CONCLUSIONS We have identified a group of adult autoimmune phenotypic type 2 diabetic patients who are Ab−T+ and thus would not be detected using autoantibody testing alone. We conclude that islet autoimmunity may be more prevalent in adult phenotypic type 2 diabetic patients than previously estimated.

Src and fibroblast growth factor 2 independently regulate signaling and gene expression induced by experimental injury to intact articular cartilage

OBJECTIVE To investigate whether cartilage injury activates protein tyrosine kinases distinct from fibroblast growth factor (FGF)-related signaling, and whether they contribute to injury-induced gene responses. METHODS Phosphokinases and protein tyrosine phosphorylation were assayed by Western blotting of cartilage lysates. Immunoprecipitation and Western blotting with 4G10 antibody and immunoprecipitation kinase assay were carried out. Tyrosine-phosphorylated proteins on silver-stained gels of injured cartilage lysates were identified by mass spectrometry. Messenger RNA induction in cartilage explants was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS Protein tyrosine phosphorylation occurred within seconds of injury to the surface of intact articular cartilage, as did activation of MAPKs and IKK. Activation did not reoccur upon reinjury of cultured explants. The prominent tyrosine-phosphorylated proteins focal adhesion kinase, paxillin, and cortactin were identified as substrates of Src family kinases. The Src family kinase inhibitor PP2 blocked injury-induced tyrosine phosphorylation. It did not prevent activation of the MAPKs and IKK but differentially inhibited 8 of 10 inflammatory response genes that were induced by injury. In contrast, FGF signaling blockade with PD173074 reduced all MAPK and IKK activation by ∼50% and inhibited a different subset of genes but had no effect on Src-like signaling. CONCLUSION Injury to the surface of intact articular cartilage activates Src-like kinases as well as MAPKs and IKK (implying NF-κB activation). FGF-2 contributes to MAPK/IKK activation but not to Src-like signaling, suggesting that the latter is a parallel pathway that also regulates the injury-induced inflammatory gene response.

Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.

Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin‐1 (IL‐1). IL‐1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL‐1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes.

Nociceptive Sensitizers Are Regulated in Damaged Joint Tissues, Including Articular Cartilage, When Osteoarthritic Mice Display Pain Behavior

Pain is the most common symptom of osteoarthritis (OA), yet where it originates in the joint and how it is driven are unknown. The aim of this study was to identify pain‐sensitizing molecules that are regulated in the joint when mice subjected to surgical joint destabilization develop OA‐related pain behavior, the tissues in which these molecules are being regulated, and the factors that control their regulation.

Identification of the general transcription factor Yin Yang 1 as a novel and specific binding partner for S6 Kinase 2

S6 kinase is a member of the AGC family of serine/threonine kinases and plays a key role in diverse cellular processes including cell growth and metabolism. Although, the high degree of homology between S6K family members (S6K1 and S6K2) in kinase and kinase-extension domains, the two proteins are highly divergent in the N- and C-terminal regulatory regions, hinting at differential regulation, downstream signalling and cellular function. Deregulated signalling via S6Ks has been linked to various human pathologies, such as diabetes and cancer. Therefore, S6K has emerged as a promising target for drug development. Much of what we know about S6K signalling in health and disease comes from studies of S6K1, as molecular cloning of this isoform was reported a decade earlier than S6K2. In this study, we report for the first time, the identification of the general transcription factor Yin Yang 1 (YY1) as a novel and specific binding partner of S6K2, but not S6K1. The interaction between YY1 and S6K2 was demonstrated by co-immunoprecipitation of transiently overexpressed and endogenous proteins in a number of cell lines, including HEK293, MCF7 and U937. Furthermore, direct association between S6K2 and YY1 was demonstrated by GST pull-down assay using recombinant proteins. A panel of deletion mutants was used to show that the C-terminal regulatory region of S6K2 mediates the interaction with YY1. Interestingly, the complex formation between S6K2 and YY1 can be detected in serum-starved cells, but the interaction is strongly induced in response to mitogenic stimulation. The induction of S6K2/YY1 complex formation in response to serum stimulation is abolished by pre-treatment of cells with the mTOR inhibitor, rapamycin. Furthermore, mTOR is also detected in complex with YY1 and S6K2 in serum-stimulated cells. We utilized size exclusion chromatography along with co-immunoprecipitation analysis to demonstrate the existence of the mTOR/S6K2/YY1 complex in high molecular weight fractions, which might also involve other cellular proteins. The physiological significance of the mTOR/S6K2/YY1 complex, which is induced in response to mitogenic stimulation, remains to be further investigated.

Glycogen storage disease type III presenting with secondary diabetes and managed with insulin: a case report.

Introduction Reports of secondary diabetes in glycogen storage disease type III have been very limited, where the pathogenesis and management have not been clear. Here we report on a rare case of secondary diabetes in glycogen storage disease type III that has been successfully managed with insulin. Case presentation This is a 19-year-old female of Egyptian ethnicity, born of a consanguineous marriage and known to have glycogen storage disease type III since the age of 2½ years. She presented to us with a history of polyuria, polydipsia, and loss of weight of a few days duration. Physical exam showed stunted growth, hepatomegaly, myopathy and mild dehydration. Emergency labs revealed a fasting blood glucose of 276 mg/dl, but with no ketonuria and arterial blood gases were essentially normal. Her liver transaminases were mildly elevated at the time. Review of her records revealed that the diagnosis of glycogen storage disease type III was made at the age of 2½ when the mother reported repeated attacks of afebrile (hypoglycemic) convulsions, increasing abdominal girth and failure to thrive. The diagnosis was confirmed by demonstration of debrancher enzyme deficiency on enzymatic assay. Over the years she developed liver dysfunction along with other complications and subsequently her hypoglycemic attacks disappeared a few years prior to her current presentation. After careful consideration of different treatment options, and considering she had been free of hypoglycemic attacks for a few years and had liver dysfunction, we chose to cautiously initiate the patient on insulin therapy. She was still poorly controlled and we gradually increased her total daily dose to 0.8 u/kg. She continued to be free of hypoglycemic attacks and her average daily blood glucose is about 160 mg/dl. Conclusion We report a rare case of secondary diabetes mellitus in a patient with glycogen storage disease type III managed with insulin. We recommend insulin therapy over oral hypoglycemics to avoid further hepatotoxicity, provided hypoglycemia has resolved. We also recommend serial follow up of glycogen storage disease type III patients with an oral glucose tolerance test for early detection and management of glucose intolerance.IntroductionReports of secondary diabetes in glycogen storage disease type III have been very limited, where the pathogenesis and management have not been clear. Here we report on a rare case of secondary diabetes in glycogen storage disease type III that has been successfully managed with insulin.Case presentationThis is a 19-year-old female of Egyptian ethnicity, born of a consanguineous marriage and known to have glycogen storage disease type III since the age of 2½ years. She presented to us with a history of polyuria, polydipsia, and loss of weight of a few days duration. Physical exam showed stunted growth, hepatomegaly, myopathy and mild dehydration. Emergency labs revealed a fasting blood glucose of 276 mg/dl, but with no ketonuria and arterial blood gases were essentially normal. Her liver transaminases were mildly elevated at the time. Review of her records revealed that the diagnosis of glycogen storage disease type III was made at the age of 2½ when the mother reported repeated attacks of afebrile (hypoglycemic) convulsions, increasing abdominal girth and failure to thrive. The diagnosis was confirmed by demonstration of debrancher enzyme deficiency on enzymatic assay. Over the years she developed liver dysfunction along with other complications and subsequently her hypoglycemic attacks disappeared a few years prior to her current presentation. After careful consideration of different treatment options, and considering she had been free of hypoglycemic attacks for a few years and had liver dysfunction, we chose to cautiously initiate the patient on insulin therapy. She was still poorly controlled and we gradually increased her total daily dose to 0.8 u/kg. She continued to be free of hypoglycemic attacks and her average daily blood glucose is about 160 mg/dl.ConclusionWe report a rare case of secondary diabetes mellitus in a patient with glycogen storage disease type III managed with insulin. We recommend insulin therapy over oral hypoglycemics to avoid further hepatotoxicity, provided hypoglycemia has resolved. We also recommend serial follow up of glycogen storage disease type III patients with an oral glucose tolerance test for early detection and management of glucose intolerance.

S6 Kinase 2 Is Bound to Chromatin-Nuclear Matrix Cellular Fractions and Is Able to Phosphorylate Histone H3 at Threonine 45 In Vitro and In Vivo

The activity of S6 kinases (S6K) is highly induced in cancer cells highlighting an essential role in carcinogenesis. The S6K family has two members: S6K1 and S6K2 which bear common as well as distinct features. In an attempt to identify S6K2 unique sequence features compared to S6K1, we applied extensive bioinformatic analysis and motif search approaches. Interestingly, we identified 14 unique protein signatures which are present in proteins directly connected to chromatin and/or involved in transcription regulation. Using chromatin binding assay, we biochemically showed that S6K2 is bound to chromatin as well as nuclear matrix cellular fractions in HEK293 cells. The presence of S6K2 in chromatin fractions raised the possibility that it may be in close proximity to a number of chromatin substrates. For that, we then searched for S6K phosphorylation consensus sites RXRXXT/S in mammalian proteins using the SWISS‐PROT database. Interestingly, we identified some potential phosphorylation sites in histone H3 (Thr45). Using in vitro kinase assays and siRNA‐based knockdown strategy; we confirmed that S6K2 but not S6K1 or AKT is essential for histone H3‐Thr45 phosphorylation in HEK293 cells. Furthermore, we show that the nuclear localisation sequence in the S6K2 C‐terminus is essential for this modification. We have found that, H3‐Thr45 phosphorylation correlates to S6K activation in response to mitogens and TPA‐induced cell differentiation of leukaemic cell lines U937, HL60 and THP1. Overall, we demonstrate that S6K2 is a novel kinase that can phosphorylate histone H3 at position Thr45, which may play a role during cell proliferation and/or differentiation. J. Cell. Biochem. 115: 1048–1062, 2014.

Brief Report: JNK-2 Controls Aggrecan Degradation in Murine Articular Cartilage and the Development of Experimental Osteoarthritis.

The pathogenesis of osteoarthritis (OA) is poorly understood. Loss of the proteoglycan aggrecan from cartilage is an early event. Recently, we identified a role for the JNK pathway, particularly JNK‐2, in human articular chondrocytes in vitro in regulating aggrecan degradation. The present study was undertaken to investigate whether JNK‐2 has a similar function in vivo and to examine its role in gene expression.

Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K63-Linked Polyubiquitination Upon Injury to Animal Articular Cartilage

Mechanical injury to cartilage predisposes to osteoarthritis (OA). Wounding of the articular cartilage surface causes rapid activation of MAP kinases and NF‐κB, mimicking the response to inflammatory cytokines. This study was undertaken to identify the upstream signaling mechanisms involved.