Targeted inhibition of ANKRD1 disrupts sarcomeric ERK-GATA4 signal transduction and abrogates phenylephrine-induced cardiomyocyte hypertrophy.

AIMS: Accumulating evidence suggest that sarcomere signalling complexes play a pivotal role in cardiomyocyte hypertrophy by communicating stress signals to the nucleus to induce gene expression. Ankyrin repeat domain 1 (ANKRD1) is a transcriptional regulatory protein that also associates with sarcomeric titin; however, the exact role of ANKRD1 in the heart remains to be elucidated. We therefore aimed to examine the role of ANKRD1 in cardiomyocyte hypertrophic signalling. METHODS AND RESULTS: In neonatal rat ventricular myocytes, we found that ANKRD1 is part of a sarcomeric signalling complex that includes ERK1/2 and cardiac transcription factor GATA4. Treatment with hypertrophic agonist phenylephrine (PE) resulted in phosphorylation of ERK1/2 and GATA4 followed by nuclear translocation of the ANKRD1/ERK/GATA4 complex. Knockdown of Ankrd1 attenuated PE-induced phosphorylation of ERK1/2 and GATA4, inhibited nuclear translocation of the ANKRD1 complex, and prevented cardiomyocyte growth. Mice lacking Ankrd1 are viable with normal cardiac function. Chronic PE infusion in wild-type mice induced significant cardiac hypertrophy with reactivation of the cardiac fetal gene program which was completely abrogated in Ankrd1 null mice. In contrast, ANKRD1 does not play a role in haemodynamic overload as Ankrd1 null mice subjected to transverse aortic constriction developed cardiac hypertrophy comparable to wild-type mice. CONCLUSION: Our study reveals a novel role for ANKRD1 as a selective regulator of PE-induced signalling whereby ANKRD1 recruits and localizes GATA4 and ERK1/2 in a sarcomeric macro-molecular complex to enhance GATA4 phosphorylation with subsequent nuclear translocation of the ANKRD1 complex to induce hypertrophic gene expression.

Global deletion of Ankrd1 results in a wound-healing phenotype associated with dermal fibroblast dysfunction.

The expression of ankyrin repeat domain protein 1 (Ankrd1), a transcriptional cofactor and sarcomeric component, is strongly elevated by wounding and tissue injury. We developed a conditional Ankrd1(fl/fl) mouse, performed global deletion with Sox2-cre, and assessed the role of this protein in cutaneous wound healing. Although global deletion of Ankrd1 did not affect mouse viability or development, Ankrd1(-/-) mice had at least two significant wound-healing phenotypes: extensive necrosis of ischemic skin flaps, which was reversed by adenoviral expression of ANKRD1, and delayed excisional wound closure, which was characterized by decreased contraction and reduced granulation tissue thickness. Skin fibroblasts isolated from Ankrd1(-/-) mice did not spread or migrate on collagen- or fibronectin-coated surfaces as efficiently as fibroblasts isolated from Ankrd1(fl/fl) mice. More important, Ankrd1(-/-) fibroblasts failed to contract three-dimensional floating collagen gels. Reconstitution of ANKRD1 by adenoviral infection stimulated both collagen gel contraction and actin fiber organization. These in vitro data were consistent with in vivo wound closure studies, and suggest that ANKRD1 is important for the proper interaction of fibroblasts with a compliant collagenous matrix both in vitro and in vivo.

ANKRD1 acts as a transcriptional repressor of MMP13 via the AP-1 site.

The transcriptional cofactor ANKRD1 is sharply induced during wound repair, and its overexpression enhances healing. We recently found that global deletion of murine Ankrd1 impairs wound contraction and enhances necrosis of ischemic wounds. A quantitative PCR array of Ankrd1(-/-) (KO) fibroblasts indicated that ANKRD1 regulates MMP genes. Yeast two-hybrid and coimmunoprecipitation analyses associated ANKRD1 with nucleolin, which represses AP-1 activation of MMP13. Ankrd1 deletion enhanced both basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP13 promoter activity; conversely, Ankrd1 overexpression in control cells decreased PMA-induced MMP13 promoter activity. Ankrd1 reconstitution in KO fibroblasts decreased MMP13 mRNA, while Ankrd1 knockdown increased these levels. MMP13 mRNA and protein were elevated in intact skin and wounds of KO versus Ankrd1(fl/fl) (FLOX) mice. Electrophoretic mobility shift assay gel shift patterns suggested that additional transcription factors bind to the MMP13 AP-1 site in the absence of Ankrd1, and this concept was reinforced by chromatin immunoprecipitation analysis as greater binding of c-Jun to the AP-1 site in extracts from FLOX versus KO fibroblasts. We propose that ANKRD1, in association with factors such as nucleolin, represses MMP13 transcription. Ankrd1 deletion additionally relieved MMP10 transcriptional repression. Nuclear ANKRD1 appears to modulate extracellular matrix remodeling by MMPs.

ALCAM/CD166 is a TGF-ß-responsive marker and functional regulator of prostate cancer metastasis to bone.

The dissemination of prostate cancer to bone is a common, incurable aspect of advanced disease. Prevention and treatment of this terminal phase of prostate cancer requires improved molecular understanding of the process as well as markers indicative of molecular progression. Through biochemical analyses and loss-of-function in vivo studies, we demonstrate that the cell adhesion molecule, activated leukocyte cell adhesion molecule (ALCAM), is actively shed from metastatic prostate cancer cells by the sheddase ADAM17 in response to TGF-ß. Not only is this posttranslational modification of ALCAM a marker of prostate cancer progression, the molecule is also required for effective metastasis to bone. Biochemical analysis of prostate cancer cell lines reveals that ALCAM expression and shedding is elevated in response to TGF-ß signaling. Both in vitro and in vivo shedding is mediated by ADAM17. Longitudinal analysis of circulating ALCAM in tumor-bearing mice revealed that shedding of tumor, but not host-derived ALCAM is elevated during growth of the cancer. Gene-specific knockdown of ALCAM in bone-metastatic PC3 cells greatly diminished both skeletal dissemination and tumor growth in bone. The reduced growth of ALCAM knockdown cells corresponded to an increase in apoptosis (caspase-3) and decreased proliferation (Ki67). Together, these data demonstrate that the ALCAM is both a functional regulator as well as marker of prostate cancer progression.

26S proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells.

Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

Disruption of a GATA4/Ankrd1 signaling axis in cardiomyocytes leads to sarcomere disarray: implications for anthracycline cardiomyopathy.

Doxorubicin (Adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s) of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA) similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4) in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent depletion of GATA4 and CARP in cardiomyocytes by doxorubicin contributes in large part to myofibrillar disarray and the overall pathophysiology of anthracycline cardiomyopathy.

MafA and MafB regulate Pdx1 transcription through the Area II control region in pancreatic beta cells.

Pancreatic-duodenal homeobox factor-1 (Pdx1) is highly enriched in islet beta cells and integral to proper cell development and adult function. Of the four conserved 5'-flanking sequence blocks that contribute to transcription in vivo, Area II (mouse base pairs -2153/-1923) represents the only mammalian specific control domain. Here we demonstrate that regulation of beta-cell-enriched Pdx1 expression by the MafA and MafB transcription factors is exclusively through Area II. Thus, these factors were found to specifically activate through Area II in cell line transfection-based assays, and MafA, which is uniquely expressed in adult islet beta cells was only bound to this region in quantitative chromatin immunoprecipitation studies. MafA and MafB are produced in beta cells during development and were both bound to Area II at embryonic day 18.5. Expression of a transgene driven by Pdx1 Areas I and II was also severely compromised during insulin+ cell formation in MafB(-/-) mice, consistent with the importance of this large Maf in beta-cell production and Pdx1 expression. These findings illustrate the significance of large Maf proteins to Pdx1 expression in beta cells, and in particular MafB during pancreatic development.

CARP: fishing for novel mechanisms of neovascularization.

Gene expression profiling of mouse skin wounds has led to the discovery of numerous target genes that may have therapeutic or diagnostic value. Among these, cardiac ankyrin repeat protein (CARP, ankrd1) expression was markedly and persistently elevated in several cutaneous compartments. This review summarizes the current state of knowledge of CARP and its regulation in biological systems. In addition to its role as a nuclear transcription cofactor in many cell types including vascular endothelium, CARP is also a structural component of the sarcomere. CARP transcripts are prominent in cardiogenesis and muscle injury, and they are under complex regulation by cytokines, hypoxia, doxorubicin, and other forms of stress. CARP overexpression in wounds by adenoviral gene transfer leads to a high vascular density, and CARP exerts effects on endothelial behavior. The unusual cellular distribution and actions of CARP make it a novel candidate gene in tissue repair.

Interactions between areas I and II direct pdx-1 expression specifically to islet cell types of the mature and developing pancreas.

PDX-1 regulates transcription of genes involved in islet beta cell function and pancreas development. Islet-specific expression is controlled by 5'-flanking sequences from base pair (bp) -2917 to -1918 in transgenic experiments, which encompasses both conserved (i.e. Area I (bp -2761/-2457), Area II (bp -2153/-1923)) and non-conserved pdx-1 sequences. However, only an Area II-driven transgene is independently active in vivo, albeit in only a fraction of islet PDX-1-producing cells. Our objective was to identify the sequences within the -2917/-1918-bp region that act in conjunction with Area II to allow comprehensive expression in islet PDX-1(+) cells. In cell line-based transfection assays, only Area I effectively potentiated Area II activity. Both Area I and Area II functioned in an orientation-independent manner, whereas synergistic, enhancer-like activation was uniquely found with duplicated Area II. Chimeras of Area II and the generally active SV40 enhancer or the beta cell-specific insulin enhancer suggested that islet cell-enriched activators were necessary for Area I activation, because Area II-mediated stimulation was reduced by the SV40 enhancer and activated by the insulin enhancer. Several conserved sites within Area I were important in Area I/Area II activation, with binding at bp -2614/-2609 specifically controlled by Nkx2.2, an insulin gene regulator that is required for terminal beta cell differentiation. The ability of Area I to modulate Area II activation was also observed in vivo, as an Area I/Area II-driven transgene recapitulated the endogenous pdx-1 expression pattern in developing and adult islet cells. These results suggest that Area II is a central pdx-1 control region, whose islet cell activity is uniquely modified by Area I regulatory factors.

The islet beta cell-enriched RIPE3b1/Maf transcription factor regulates pdx-1 expression.

Pancreatic duodenal homeobox factor-1, PDX-1, is required for pancreas development, islet cell differentiation, and the maintenance of beta cell function. Selective expression in the pancreas appears to be principally regulated by Area II, one of four conserved regulatory sequence domains found within the 5'-flanking region of the pdx-1 gene. Detailed mutagenesis studies have identified potential sites of interaction for both positive- and negative-acting factors within the conserved sequence blocks of Area II. The islet beta cell-enriched RIPE3b1 transcription factor, the activator of insulin C1 element-driven expression, was shown here to also stimulate Area II by binding to sequence blocks 4 and 5 (termed B4/5). Accordingly, B4/5 DNA-binding protein's molecular mass (i.e. 46 kDa), binding specificity, and islet beta cell-enriched distribution were identical to RIPE3b1. Area II-mediated activation was also unaffected upon replacing B4/5 with the insulin C1/RIPE3b1 binding site. In addition, the chromatin immunoprecipitation assay showed that the Area II region of the endogenous pdx-1 gene was precipitated by an antiserum that recognizes the large Maf protein that comprises the RIPE3b1 transcription factor. These results strongly suggest that RIPE3b1/Maf has an important role in generating and maintaining physiologically functional beta cells.

Conserved sequences in a tissue-specific regulatory region of the pdx-1 gene mediate transcription in Pancreatic beta cells: role for hepatocyte nuclear factor 3 beta and Pax6.

Pancreas duodenum homeobox 1 (PDX-1) is absolutely required for pancreas development and the maintenance of islet beta-cell function. Temporal and cell-type-specific transcription of the pdx-1 gene is controlled by factors acting upon sequences found within its 5'-flanking region. Critical cis-acting transcriptional control elements are located within a nuclease hypersensitive site that contains three conserved subdomains, termed areas I, II, and III. We show that area II acts as a tissue-specific regulatory region of the pdx-1 gene, directing transgene expression to a subpopulation of islet cells. Mutation of the area II hepatocyte nuclear factor 3 (HNF3) binding element in the larger area I- and area II- containing PstBst fragment also decreases PB(hsplacZ) transgene penetrance. These two results indicate possible ontogenetic and/or functional heterogeneity of the beta-cell population. Several other potential positive- and negative-acting control elements were identified in area II after mutation of the highly conserved sequence blocks within this subdomain. Pax6, a factor essential for islet alpha-cell development and islet hormone gene expression, was shown to bind in area II in vitro. Pax6 and HNF3 beta were also found to bind to this region in vivo by using the chromatin immunoprecipitation assay. Collectively, these data suggest an important role for both HNF3 beta and Pax6 in regulating pdx-1 expression in beta cells.