Growth Hormone, Stat5b, and IGF-I Transcriptional Circuit

Mapping the Growth Hormone–Stat5b–IGF-I Transcriptional Circuit (Rotwein, 2012)

Introduction: The GH, IGF-I, and Stat5b Circuit

• Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are potent regulators of:

  • Somatic growth

  • Metabolism

  • Tissue repair

• They have also been implicated in aging and carcinogenesis.

• The intimate link between GH and IGF-I was established over $50$ years ago with the somatomedin hypothesis.

• Recent studies confirm that GH strongly stimulates IGF-I gene transcription, thereby controlling IGF-I production.

• A crucial mediator in the GH–IGF-I biosynthetic pathway is the latent transcription factor Stat5b.

• This review explores the complex mechanistic relationship between GH action, Stat5b, and IGF-I gene activation.

• It suggests Stat5b has a broad role in mediating IGF-I gene regulation in response to various physiological inputs.

Historical Perspective: The GH–IGF-I–Stat5b Connection

• Early Discoveries ($\text{>50}$ years ago):

  • Biochemical mechanisms of GH action were unknown.

  • Studies on GH's acute effects on protein synthesis in rib cartilage explants (measured by sulfate incorporation into proteoglycans) led to the somatomedin hypothesis.

  • This hypothesis proposed that GH promotes growth through a mediator molecule, a 'somatomedin' [1,2].

  • Key experiments showed GH was inactive directly on cartilage, but 'sulfation factor' activity appeared in serum within a few hours of systemic GH administration to pituitary-ablated rats [1].

• Identification of IGF-I:

  • The GH-dependent somatomedin was later identified as IGF-I, a $70$-amino acid single-chain peptide [3].

  • Direct confirmation of the somatomedin hypothesis showed GH rapidly increased circulating IGF-I levels [4].

• Molecular Biological Insights (mid-$\text{1980s}$-early $\text{1990s}$):

  • Experiments determined that GH induced IGF-I gene expression [5–7].

  • A single systemic pulse of GH to pituitary-deficient rats stimulated IGF-I gene transcription within minutes and led to sustained IGF-I mRNA production [8].

• Role of Stat5b:

  • Crucial insights stemmed from characterizing the GH receptor (GHR) and its intracellular signaling cascades [9–11].

  • These studies established that Stat5b is the essential transducer of GH-stimulated IGF-I gene activation [11,12].

• Clinical Confirmation ('experiments of nature'):

  • Inactivating mutations in the $STAT5B$ gene were found in individuals with GH-resistant growth defects and low circulating IGF-I levels, confirming Stat5b's critical role [13].

• Review Focus: To discuss the molecular and biochemical basis of IGF-I gene regulation by GH via Stat5b, emphasizing a potentially complex relationship and a broader role for Stat5b beyond GH-activated pathways.

The GH Receptor (GHR) and GH-Mediated Signaling Pathways

• The GHR was one of the first cytokine receptor family members to have its biochemical signaling mechanism identified [9].

• GHR Structure and Activation (Figure 1):

  • In the absence of hormone, GHR, a single-chain membrane-spanning protein, exists primarily as a homodimer [10,11].

  • A single GH molecule binds sequentially to sites on the extracellular part of each receptor monomer; binding to the first facilitates interaction with the second [10,11].

  • GH-induced conformational changes in the receptor dimer transmit information across the cell membrane to the intracellular part of the GHR.

  • This leads to activation of the receptor-associated tyrosine protein kinase, Jak2.

  • Jak2 then phosphorylates multiple tyrosine residues within the intracellular region of the GHR [9–11].

• Stat5b Docking: Each phosphorylated tyrosine on the GHR can serve as a docking site for signaling proteins, with several sites specifically binding Stat5b (Figure 1).

Stat5b: A Crucial Mediator for GH-Stimulated Somatic Growth

• Despite multiple signaling pathways induced by ligand-activated GHR [9–11], Stat5b is the crucial signaling intermediate for GH-regulated growth [10,14,15].

• Stat Family: Stat5b is one of seven members in mammals of the Stat family of latent transcription factors [12,16].

• Early Evidence (1997): Studies showed that an engineered hypomorphic mutant of Stat5b in mice led to diminished postnatal growth [17,18].

• GHR Manipulations in Mice:

  • Elegant experiments identified a cohort of tyrosine residues within the GHR's intracellular domain crucial for Stat5b binding and activation by GHR-associated Jak2 [19] (Figure 1).

  • Eliminating all Stat5b binding sites in the GHR or mutating the Jak2 binding domain resulted in:

    • Loss of Stat5b activation in response to GH.

    • Growth deficits in mice as severe as those seen with targeted loss of the GHR [19,20].

• Human Clinical Evidence: The crucial role of Stat5b was strengthened by the discovery of individuals with profound growth failure, short stature, and loss of GH responsiveness due to mutations in the $STAT5B$ gene, which either eliminated Stat5b protein production or rendered it inactive [13].

Molecular Anatomy and Physiology of the IGF-I Gene

• Gene Structure (Figure 2):

  • In mammals, the IGF-I gene comprises six exons and five introns, spanning $>80$ kb of chromosomal DNA [21].

  • Tandem promoters (Promoter 1 and Promoter 2) direct IGF-I gene transcription through unique leader exons.

    • Promoter 1 utilizes heterogeneous transcription initiation sites and is active in multiple animal tissues [22].

    • Promoter 2 is smaller, simpler, and predominantly expressed in the liver, though not exclusively [22].

  • The biochemical mechanisms for tissue-specific promoter activity are unknown.

  • DNA sequences of both proximal promoters are relatively well-conserved in mammals (e.g., $74\%$ over $420$ nucleotides for P1; $58\%$ over $404$ nucleotides for P2 between rat and human IGF-I), suggesting conserved functional properties during speciation.

• mRNA Processing:

  • IGF-I mRNA undergoes alternative splicing involving exons $5$ and $6$, and differential polyadenylation at the $3'$ end of exon $6$ [21,22].

  • These mechanisms generate over $100$ distinct IGF-I mRNAs [21].

  • Evolutionary Pressures: The reasons for maintaining so many distinct transcripts for a peptide encoding one of two protein precursors (IGF-IA, IGF-IB) and the same $70$-amino acid mature IGF-I peptide remain unclear [21,23].

  • Hypotheses for Multiple Transcripts:

    • Each class of IGF-I transcripts might have unique properties, such as distinct half-lives.

    • A range of mRNA turnover rates could ensure sustained IGF-I protein production after gene induction, possibly through cohorts of mRNAs insensitive to specific microRNAs or inhibitory molecules.

    • Current data are fragmentary; average half-life exceeds several hours (range 4-16\ hr) [24,25], and targeting microRNAs are not elucidated.

  • Protein Precursors: Key evolutionary constraints might act at the level of the two protein precursors, IGF-IA and IGF-IB, which differ in their C-terminal sequences but are processed into the same $70$-amino acid bioactive IGF-I peptide [21,23].

• GH-Induced Transcription: The activated GH receptor rapidly stimulates the activity of both IGF-I promoters and promotes the accumulation of all classes of IGF-I mRNAs (at least in pituitary-deficient rats) [8,27].

  • In the liver, GH-induced transcription of Promoter 1 is several-fold higher than Promoter 2 [27].

  • This indicates that both promoters are targets of acute GH-mediated signaling, and all IGF-I transcripts are produced as a consequence of GH action.

Defining a Central Role for Stat5b in GH-Activated IGF-I Gene Transcription

• Initial Evidence (2003): Studies directly linked Stat5b to GH-activated GHR signaling leading to IGF-I gene transcription [28].

  • A dominant-negative Stat5b blocked GH-induced IGF-I gene transcription in pituitary-deficient rats.

  • A constitutively active Stat5b promoted IGF-I gene activation even without GH signaling [28].

• Non-Canonical Binding Sites: Unlike other GH-activated genes (e.g., murine $Cish, Socs2, Igfals, Spi2.1$) where Stat5b binds directly to proximal promoters [29], the IGF-I gene lacks canonical Stat5 binding sites in its promoters.

• Dispersed Binding Domains (Figure 3): Bioinformatic and chromatin immunoprecipitation (ChIP) experiments revealed multiple Stat5b binding domains dispersed throughout the human, rat, and mouse IGF-I loci, but not within either promoter [30–35].

  • DNA sequence analysis showed $7$ out of $10$ identified elements contained at least two recognizable Stat5b binding sites.

  • The consensus sequence for Stat5b binding is $5'-TTCNNNGAA-3'$ (top strand; N = G, A, T, or C) [33–35], previously found to preferentially bind Stat5 [36,37].

  • The remaining three elements had one single site or a non-consensus sequence [33–35].

  • Paired binding sequences are common in other Stat5b-regulated genes' proximal promoters (e.g., mouse and rat $Cish, Socs2, Igfals, Spi2.1$) [29]. Paired sequences are thought to facilitate stronger Stat-DNA interactions and enhance transcriptional information transfer [16].

  • Whole-genome ChIP-seq analyses for Stat1 showed $>75\%$ of chromosomal binding elements contained consensus sequences, with half being paired sites [38].

  • Future comprehensive and unbiased evaluations are needed as current bioinformatics focused on conserved and paired sequences for ChIP assays [33–35].

• Binding Affinity and Transcriptional Potency:

  • Stat5b binds directly to each consensus DNA sequence within putative transcriptional response elements, with variable affinities depending on central and flanking nucleotides [33,35].

  • When these Stat5b-binding elements were fused to IGF-I promoters $1$ or $2$, they enhanced GH-activated gene transcription in cell-based reporter assays [35].

  • However, transcriptional potency varied significantly and did not closely correlate with the binding affinity of individual Stat5b consensus sites [35].

• Unique Paradigm:

  • These data support that GH-induced IGF-I gene transcription is mediated by Stat5b binding to multiple dispersed conserved DNA elements in chromatin.

  • This differs from other GH- and Stat5b-regulated genes where paired Stat5b binding domains are typically in the proximal promoter region [29].

  • This paradigm appears unique among all Stat-regulated genes, as no other examples exist where multiple dispersed enhancers binding the same transcription factor exert combinatorial control over a single target gene.

GH Action and the Chromatin Landscape of the IGF-I Gene

• Active Chromatin Characteristics [40]:

  • Chromatin around active genes is typically 'open'.

  • This is reflected by histone modifications, including high levels of lysine acetylation of core histones H3 and H4, and trimethylation of lysine $4$ of histone H3.

  • These changes facilitate access of promoter DNA to RNA polymerase II and associated factors.

  • Inactive promoters typically lack these modifications.

• IGF-I Promoter Chromatin (Figure 4):

  • Surprisingly, in hepatic chromatin of young adult pituitary-deficient male rats, the two IGF-I promoters appear to reside in an open environment, even though IGF-I gene transcription is minimal without GH signaling [27].

  • The biochemical mechanisms for this are unknown; possibly due to prior GH/pituitary trophic hormone presence during early postnatal development [27].

• GH-Induced Changes:

  • Despite the already open state, GH causes a dramatic and rapid increase in core histone acetylation levels.

  • A $20$-fold rise is detected at the two IGF-I promoters within $60$ minutes of systemic hormone administration, accompanied by IGF-I gene transcription induction [27].

  • The histone acetyl-transferase (HAT) and transcriptional coactivator p300 may contribute, as it is detected at relatively high levels at IGF-I Promoter 1.

  • However, p300 is less abundant at Promoter 2 [27] and other GH- and Stat5b-activated genes (e.g., $Cish, Socs2, Igfals, Spi2.1$) [29].

  • This suggests that other, unidentified HATs might be responsible for these latter modifications, indicating potentially distinct biochemical regulatory mechanisms for acute GH-mediated chromatin changes at each IGF-I promoter and other GH-stimulated genes.

Mechanisms of Action of Stat5b in Promoting IGF-I Gene Transcription

• Chromosomal Enhancer Properties [41–43]:

  • Recent studies define transcriptional enhancer properties, often using Stat1 activation by interferon-$\gamma$ as a test case [38,42,44].

  • Binding of Stat1 to chromosomal DNA is often accompanied by:

    • Mono-methylation of lysine $4$ on histone H3 (H3K4me1).

    • Binding of p300.

    • Binding of Med1 (TRAP220, a component of the transcriptional Mediator complex [45]).

    • Binding of RNA polymerase II.

  • DNA segments with these chromatin properties can boost neutral promoter activity in cell-based assays [41].

• Stat5b Elements as Enhancers (Figure 3): At least four GH-mediated Stat5b-binding elements in the IGF-I locus exhibit a similar chromatin signature [35].

• Unresolved Questions via Chromatin Conformation Capture ($\text{3C}$) Studies:

  • To be a true enhancer, a distal element must physically interact with its cognate gene promoter in chromatin, which is the hypothesized mechanism for information transfer and transcription activation [46,47].

  • The 3C assay [48,49] assesses these transient interactions between genomic regions in chromatin, mediated by interacting proteins bound to DNA.

  • $3C$ studies have not yet been reported for the IGF-I gene, so it is unclear which GH-induced Stat5b-binding elements are true transcriptional enhancers versus 'decoys' that bind Stat5b and sequester it from activating sites.

  • The 'decoy' idea implies that some Stat5b binding elements might modify GH-activated, Stat5b-dependent transcription by titrating available Stat5b from more productive interactions.

  • Further $3C$ and experimental approaches are needed to establish how GH-induced Stat5b-binding chromosomal elements individually and collectively control IGF-I gene transcription.

• Role of Bcl6 as a Transcriptional Repressor:

  • Bcl6 is a transcriptional repressor whose DNA recognition sequence resembles that of Stat5b [50,51].

  • ChIP studies show Bcl6 bound to the Stat5b element in proximal $Socs2, Cish,$, and $Spi2.1$ promoters in the absence of GH signaling [29,52,53].

  • GH treatment leads to rapid replacement of Bcl6 with Stat5b at these sites [29,52,53].

  • GH also inhibits $Bcl6$ gene transcription via Stat5b, causing a decline in Bcl6 protein [52,53].

  • Proposed Dual Mechanism: GH may enhance transcription of some Stat5b-regulated genes by:

    1. Reducing the amount of the Bcl6 repressor.

    2. Direct Stat5b-mediated transcriptional activation.

  • However, the role of Bcl6 in the IGF-I locus is more complex: Bcl6 is found at some Stat5b binding elements, but GH treatment does not consistently promote its disappearance from these sites, even though IGF-I gene transcription is induced [29].

Stat5b: A Potential Mediator of Other Pathways Regulating IGF-I Gene Expression

• The GH–IGF-I growth pathway is modulated by diverse factors, including nutritional/metabolic perturbations, hormonal/growth factor signaling, and acute/chronic diseases [54–61]. Stat5b may thus be regulated by these processes, modifying IGF-I gene activity.

• Nutritional Deficiencies and Metabolic Deprivation:

  • Impaired somatic growth linked to nutritional deficiencies and metabolic defects [58,61].

  • Fasting leads to a decline in serum IGF-I (reversed by re-feeding) and decreased hepatic IGF-I mRNA levels (reversible) [58,62].

  • Biochemical mechanisms are multifactorial, involving reduced IGF-I gene transcription rates and mRNA stability [58].

  • Fasting promotes functional resistance to GH, seen as a decline in GH-stimulated Jak2 and Stat5b activation [62], which explains negative impacts on IGF-I gene activity [58,63].

  • FGF21 (Fibroblast Growth Factor 21):

    • A metabolic hormone implicated as a mediator of severe food deprivation [64,65].

    • Overexpression in mice inhibits IGF-I gene expression and blunts somatic growth by targeting Stat5b, reducing its tyrosine phosphorylation [66].

    • FGF21 may also participate in a GH-activated negative feedback loop, as its expression is indirectly induced by GH's acute metabolic actions on adipocytes [67].

    • This suggests FGF21 via Stat5b might mediate part of the negative impact of nutritional deficiencies on somatic growth.

• Chronic Inflammatory Diseases:

  • Some cytokines impair growth through negative effects on the GH–IGF-I pathway [59,60].

  • Interleukin-6 (IL-6): Elevated in active Crohn’s disease [60]. IL-6 stimulates suppressor of cytokine signaling protein 3 (SOCS-3), which negatively regulates the GH receptor and Jak2, thereby reducing IGF-I gene expression [60].

  • Tumor Necrosis Factor-$\alpha$ (TNF-$\alpha$): Also elevated in chronic inflammatory diseases [60]. Its signaling pathways disrupt GH-mediated Jak2 and Stat5b activation and can counter Stat5b's transcriptional effects [59,68].

  • Multiple cytokines can thus collaboratively perturb the GH–Stat5b–IGF-I transcriptional circuit.

• Glucocorticoids (GCs):

  • Excess GCs interfere with the GH–IGF-I axis at many levels [61].

  • One mechanism is GH resistance, likely due to a decline in GH receptor abundance [58].

  • The glucocorticoid receptor (GR), a steroid-activated transcription factor [69], mediates this inhibitory effect, but surprisingly, its actions are also required for normal GH- and IGF-I-mediated growth [70].

  • Targeted GR deficiency in the liver causes growth deficits in mice similar to liver-specific knockout of Stat5a and Stat5b, with a comparable decline in IGF-I gene expression [71].

  • GR interacts with Stat5b in hepatic chromatin at one of the GH-activated Stat5b-binding elements in the IGF-I locus [72].

  • Under physiological conditions, GR may act as a selective transcriptional cofactor for Stat5b on coregulated genes, potentially including IGF-I [71].

• Estrogens:

  • Estrogen effects are mediated by estrogen receptors (ER), nuclear proteins related to GR [73,74].

  • Estrogen actions on uterine proliferation appear mediated by IGF-I.

  • ER$\alpha$ (one of two ERs in mammals [73,74]) stimulates IGF-I production in uterine epithelium and stroma by inducing IGF-I gene transcription [75].

  • This involves two transcriptional mechanisms:

    1. Direct ER$\alpha$ binding: ER$\alpha$ binds directly to several sites in IGF-I chromatin (mapped to $5'$ to IGF-I promoter $1$ and intronic region between exons $3$ and $4$) [75].

    2. Induction of Stat5a expression: Stat5a then binds to at least one of the Stat5b elements in the IGF-I locus, activating IGF-I gene expression via mechanisms similar to Stat5b [75].

  • This similarity is expected, as Stat5a and Stat5b are $>96\%$ identical in amino acid sequence [12] and recognize the same core DNA binding site [36,37].

  • It is not yet established if the ER$\alpha$–Stat5a pathway is unique to the uterus or active in other tissues (e.g., mammary gland, skeleton) where estrogens promote IGF-I expression [75–78].

Concluding Remarks

• The understanding of GH's control over IGF-I biosynthesis and somatic growth has significantly advanced in the past decade.

• Stat5b is the crucial mediator, activated by Jak2 on the hormone-stimulated GH receptor.

• Active Stat5b interacts with multiple binding sites in the IGF-I locus within chromatin.

• Through currently uncharacterized mechanisms, it promotes rapid information transmission to the two IGF-I promoters, leading to IGF-I gene transcription and production of IGF-I mRNAs and protein.

• This GH–Stat5b–IGF-I transcriptional pathway's framework is defined by biochemical, molecular biological, and molecular genetic studies.

• Its medical significance is confirmed by inactivating Stat5b mutations in children with GH-resistant growth failure [13] and by evidence that many growth modifiers target components of this pathway.

• These advances drive new fundamental mechanistic questions to decipher the complexities of GH-mediated IGF-I gene regulation in both physiological and pathological conditions.

• Answers to these questions will also offer insights into other regulatory pathways controlling GH and IGF-I, which are central to human growth, tissue repair, aging, and neoplasia [79–82].

Outstanding Questions (Box 1 in Transcript)

1. How does GH-activated Stat5b facilitate communication between distal hormone response elements in chromatin and the two IGF-I promoters?

2. What are the physiological roles of individual Stat5b-binding GH-regulated chromosomal elements in GH-stimulated IGF-I gene transcription?

3. Does Stat5b play a role in repressing IGF-I gene transcription in the absence of GH-activated signaling?

4. How does Stat5b integrate inputs from multiple physiological and pathological sources that impinge upon IGF-I gene regulation?